Patent Publication Number: US-2022226809-A1

Title: Integrated purification and measurement of dna methylation and co-measurement of mutations and/or mrna expression levels in an automated reaction cartridge

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. non-provisional application Ser. No. 15/839,731, filed Dec. 12, 2017, which claims the benefit of prior U.S. provisional application No. 62/433,165, filed Dec. 12, 2016, each of which is hereby incorporated by reference in its entirety. 
    
    
     INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 27, 2022, is named CPHDP011C1US_SL.txt and is 104,271 bytes in size. 
     BACKGROUND 
     The genomes of higher eukaryotes contain the modified nucleoside 5-methyl cytosine (5-meC). This modification is usually found as part of the dinucleotide CpG in which cytosine is converted to 5-methylcytosine in a reaction that involves flipping a target cytosine out of an intact double helix and transfer of a methyl group from S-adenosylmethionine by a methyltransferase enzyme (see, e.g., Klimasauskas et al. (1994) Cell 76: 357-369). This enzymatic conversion is the primary epigenetic modification of DNA known to exist in vertebrates and is essential for normal embryonic development (see, e.g., Bird (1992)  Cell  70: 5-8; Laird and Jaenisch (1994)  Human Mol. Genet.  3: 1487-1495; and Li et al. (1992)  Cell  69: 915-926). 
     In eukaryotes, DNA methylation regulates normal cellular processes such as genomic imprinting, chromosomal instability, and X-chromosome inactivation. Typically, DNA methylation occurs at the fifth carbon position of cytosine at dinucleotide 5′-CpG-3′ sites in or near gene promoters termed CpG islands or shores. Methylation controls gene expression by down-regulating transcription either by directly inhibiting transcriptional machinery or indirectly through the recruitment of chromatin remodeling proteins. Chromosomal methylation patterns change dynamically during embryonic development, and the correct methylation patterns have to be maintained throughout an individual&#39;s lifetime. Changes in methylation patterns are linked to aging, and errors in DNA methylation are among the earliest changes that occur during oncogenesis. Thus, the detection of methylation at gene promoters is important, inter alia, for diagnosing and/or monitoring patients with cancer. 
     Epigenetic alterations, including DNA methylation, interrupt the DNA-RNA-protein axis which describes how genetic information is transcribed into messenger RNAs (mRNAs). The correlation between genomic DNA variation, mRNA copy numbers and protein levels may be described by DNA methylation levels. Thus co-measurement of DNA methylation levels and corresponding down-stream mRNA levels can be important to understanding the mechanism of epigenetic cellular regulation. 
     Several methods have been developed to detect and quantify methylation efficiently and accurately. The most common technique is the bisulfite conversion method which converts unmethylated cytosines to uracil. Once converted, the methylation profile of DNA can be determined by standard PCR techniques, sequencing methods, and the like. 
     There are several DNA Methylation kits suitable for bisulfite conversion and DNA cleanup (e.g., EZ DNA METHYLATION™ kits from Zymo Research). Most kits involve several steps, reagents, and incubation times and often require purified DNA before conversion although some kits can utilize tissue or plasma/serum as starting material. 
     Typically the bisulfite conversion process requires at least four steps: 1) DNA Denaturation; 2) Bisulfite Incubation; 3) DNA Purification; and 4) Desulphonation. The final desulphonation step can be completed on-column or in solution followed by an ethanol precipitation. There are currently no methylation kits that allow a user to complete the entire process-DNA purification, bisulfite incubation, desulphonation, second DNA purification, and methylation-specific PCR all in one step. 
     SUMMARY 
     Various embodiments contemplated herein may comprise, but need not be limited to, one or more of the following: 
     Various embodiments contemplated herein may include, but need not be limited to, one or more of the following: 
     Embodiment 1: A method of determining the methylation state of a nucleic acid, said method comprising: 
     i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and/or filters nucleic acids in said sample and thereby purifies the DNA; 
     ii) eluting the bound DNA from the first matrix material and denaturing the DNA to produce eluted denatured DNA; 
     iii) heating the eluted DNA in the presence of a bisulfite reagent to produce a deaminated nucleic acid; 
     iv) contacting said deaminated nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material; 
     v) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a converted (e.g., bisulfite converted) nucleic acid; 
     vi) eluting said bisulfite converted nucleic acid from said second matrix material; and 
     vii) performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said converted nucleic acid to determine the methylation of said nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge. 
     Embodiment 2: The method of embodiment 1, wherein at least steps iv) through vi) are performed in a single reaction cartridge. 
     Embodiment 3: The method of embodiment 1, wherein at least steps iii) through vi) are performed in a single reaction cartridge. 
     Embodiment 4: The method of embodiment 1, wherein at least steps ii) through vi) are performed in a single reaction cartridge. 
     Embodiment 5: The method of embodiment 1, wherein at least steps i) through vi) are performed in a single reaction cartridge. 
     Embodiment 6: The method according to any one of embodiments 1-5, wherein step vii is performed in the same reaction cartridge. 
     Embodiment 7: The method according to any one of embodiments 1-6, wherein said first matrix material and said second matrix material are the same material forming the same column. 
     Embodiment 8: The method according to any one of embodiments 1-7, wherein said first matrix material and said second matrix material form different columns. 
     Embodiment 9: The method according to any one of embodiments of embodiment 1-8, wherein said methylation specific PCR, when performed, is performed in said cartridge. 
     Embodiment 10: The method according to any one of embodiments 1-9, wherein said nucleic acid sequencing, when performed, is performed in said cartridge or in a device coupled to said cartridge. 
     Embodiment 11: The method according to any one of embodiments 1-10, wherein said cartridge comprises a column comprising said first matrix material, a sample receiving chamber, a temperature controlled channel or chamber, a plurality of chambers containing reagents and/or buffers, and when in use at least one of said chambers contains a desulphonation/elution buffer, and wherein said cartridge optionally comprises a second column comprising said second matrix material. 
     Embodiment 12: The method of embodiment 11, wherein, when in use, at least one of said chambers contains a reagent that provides bisulfite ions. 
     Embodiment 13: The method according to any one of embodiments 11-12, wherein said second column is absent. 
     Embodiment 14: The method according to any one of embodiments 11-13, wherein said second column is present. 
     Embodiment 15: The method according to any one of embodiments 11-14, wherein said cartridge comprises a thermocycling channel or chamber in addition to said temperature controlled channel or chamber. 
     Embodiment 16: The method according to any one of embodiments 11-14, wherein said temperature controlled channel or chamber is a thermocycling channel or chamber. 
     Embodiment 17: The method according to any one of embodiments 11-16, wherein said cartridge comprises one or more chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer. 
     Embodiment 18: The method of embodiment 17, wherein said cartridge comprises one or more chambers containing one or more primers and probes for detection of methylation of a forward strand of a bisulfate-converted DNA. 
     Embodiment 19: The method according to any one of embodiments 17-18, wherein said cartridge comprises one or more chambers containing one or more primers and probes for detection of methylation of a reverse strand of a bisulfate-converted DNA. 
     Embodiment 20: The method according to any one of embodiments 11-19, wherein said sample receiving chamber, said column(s), said plurality of chambers, and when present, said temperature controlled channel or chamber and/or thermocycling channel or chamber, are selectively in fluid communication. 
     Embodiment 21: The method of embodiment 20, wherein said sample receiving chamber, said column(s), said plurality of chambers, and when present, said thermocycling channel or chamber, are selectively in fluid communication by microfluidic channels and valves. 
     Embodiment 22: The method of embodiment 20, wherein said sample receiving chamber, said column(s), said plurality of chambers, and when present, said thermocycling channel or chamber or a port into said thermocycling channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve. 
     Embodiment 23: The method according to any one of embodiments 11-22, wherein said cartridge, when in use, comprises: 
     a first chamber containing a sample; 
     a second chamber containing a guanidinium thiocyanate-ethanol (GTC-EtOH) solution; 
     a third chamber containing a bisulfate reagent; 
     a fourth chamber containing a buffer; 
     a fifth chamber containing a rinse solution; and 
     a sixth chamber containing an elution/desulphonation reagent. 
     Embodiment 24: The method of embodiment 23, wherein first chamber contains said sample in a GTC-EtOH-Tween extraction/precipitation reagent. 
     Embodiment 25: The method according to any one of embodiments 23-24, wherein the GTC-ETOH-Tween buffer is added at or near the time the sample is placed into the cartridge. 
     Embodiment 26: The method according to any one of embodiments 23-25, wherein the bisulfite reagent is added to the cartridge at or near the time the sample is placed in the cartridge. 
     Embodiment 27: The method of embodiment 23, wherein the GTC-ETOH-Tween buffer is provided as a component of the cartridge. 
     Embodiment 28: The method according to any one of embodiments 23-25, wherein the bisulfite reagent is provided as a component of the cartridge. 
     Embodiment 29: The method according to any one of embodiments 11-28, wherein said cartridge comprises a seventh chamber containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 30: The method according to any one of embodiments 11-29, wherein said cartridge comprises an eighth chamber also containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 31: The method of embodiments 29-30, wherein said PCR primers, and/or probes, and/or enzymes are provided as beads. 
     Embodiment 32: The method according to any one of embodiments 1-31, wherein said biological sample comprises one or more samples selected from the group consisting of a cell, a tissue, and a biological fluid containing a nucleic acid. 
     Embodiment 33: The method of embodiment 32, wherein said biological sample comprises a biological fluid selected from the group consisting of whole blood, plasma, serum, saliva, mucus, urine, sputum, pancreatic juice, and cerebrospinal fluid. 
     Embodiment 34: The method of embodiment 32, wherein said biological sample comprises a sample selected from the group consisting of a tissue sample, a formalin fixed paraffin embedded (FFPE) tissue, fresh frozen tissue, fine needle aspirates (FNA), and a core biopsy. 
     Embodiment 35: The method according to any one of embodiments 1-34, wherein said method comprises contacting said biological sample with a lysis solution. 
     Embodiment 36: The method of embodiment 35, wherein said method comprises providing said sample in said sample receiving chamber and contacting said sample with an extraction/precipitation solution. 
     Embodiment 37: The method according to any one of embodiments 1-36, wherein said matrix material comprises a column material selected from the group consisting of glass or silica, an ion exchange resin, cellulose, and hydroxyapatite. 
     Embodiment 38: The method of embodiment 37, wherein said matrix material comprises glass. 
     Embodiment 39: The method according to any one of embodiments 1-38, wherein said bisulfite ion is provided as compound selected from the group consisting of ammonium bisulfite, sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO. 
     Embodiment 40: The method of embodiment 39, wherein said bisulfite ion is provided by ammonium bisulfite. 
     Embodiment 41: The method according to any one of embodiments 1-40, wherein said bisulfite is provided in a reagent mix comprising scavengers to prevent sulfite oxidation and/or catalysts. 
     Embodiment 42: The method of embodiment 41, wherein said bisulfite is provided in a reagent mix comprising scavengers selected from the group consisting of TROLOX water-soluble analog of vitamin E and hydroquinone. 
     Embodiment 43: The method according to any one of embodiments 41-42, wherein said bisulfite is provided in a reagent mix comprising polyamines as catalysts. 
     Embodiment 44: The method according to any one of embodiments 1-43, wherein said eluting the bound DNA comprises eluting and denaturing said DNA using a low concentration of potassium hydroxide or other base. 
     Embodiment 45: The method of embodiment 44, wherein said eluting the bound DNA comprises eluting and denaturing said DNA with an alkaline solution with a pH greater than about pH 10.5. 
     Embodiment 46: The method of embodiment 44, wherein said eluting the bound DNA comprises eluting and denaturing said DNA with an alkaline solution with a pH greater than about pH 12. 
     Embodiment 47: The method of embodiments 45-46, wherein said alkaline solution is a 10-15 mM KOH solution. 
     Embodiment 48: The method according to any one of embodiments 1-47, wherein said incubating the eluted DNA with bisulfite ions to produce a deaminated nucleic acid comprises incubating the DNA in an ammonium bisulfite solution having a concentration that ranges from about 6M to about 7M. 
     Embodiment 49: The method of embodiment 48, wherein said incubating the eluted DNA with bisulfite ions to produce a deaminated nucleic acid comprises incubating the DNA in an ammonium bisulfite solution having a concentration of about 6.5M. 
     Embodiment 50: The method of embodiment 49, wherein said incubating comprises transferring the DNA in a concentrated bisulfite solution into a temperature controlled channel or chamber in said cartridge and heating said mixture. 
     Embodiment 51: The method of embodiment 50, wherein said incubating comprises thermally cycling the concentrated bisulfite solution from a temperature of about 60° C. to about 95° C. 
     Embodiment 52: The method according to any one of embodiments 1-51, wherein said contacting said deaminated nucleic acid to a second matrix material comprises mixing the DNA-bisulfite solution with fresh GTC-EtOH and dispensing the solution over said second matrix material. 
     Embodiment 53: The method of embodiment 52, wherein said method comprises washing the DNA in said second matrix material with fresh GTC-EtOH, and then a rinse solution. 
     Embodiment 54: The method of embodiment 53, wherein said rinse solution comprises PEG200. 
     Embodiment 55: The method according to any one of embodiments 1-54, wherein said desulphonating the bound deaminated nucleic acid comprises eluting the DNA from said second column with a high pH desulphonation buffer and incubating said solution. 
     Embodiment 56: The method of embodiment 55, wherein said incubating is for a period of time ranging from about 1 minute to about 1 hour, or from about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes, or for about 15 minutes. 
     Embodiment 57: The method of embodiments 55-56, wherein said high pH desulphonation/elution buffer comprises KOH. 
     Embodiment 58: The method according to any one of embodiments 55-57, wherein said incubation is in a chamber that previously held said high pH desulphonation buffer (e.g., chamber 10). 
     Embodiment 59: The method according to any one of embodiments 1-58, wherein after the incubation with bisulfite ions, a temperature controlled channel or chamber is washed with a buffer to remove the residual bisulfite and neutralize pH. 
     Embodiment 60: The method according to any one of embodiments 1-59, wherein high resolution melting analysis (HRM) on said bisulfite-converted nucleic acid is performed to determine the methylation of said nucleic acid. 
     Embodiment 61: The method according to any one of embodiments 1-60, wherein nucleic acid sequencing of said bisulfite-converted nucleic acid is performed to determine the methylation of said nucleic acid. 
     Embodiment 62: The method according to any one of embodiments 1-60, wherein methylation specific PCR is performed to determine methylation of target nucleic acid sequences. 
     Embodiment 63: The method of embodiment 62, wherein said methylation specific PCR (MSP) is performed using primers specific for methylated sequences and/or primers specific for unmethylated sequences. 
     Embodiment 64: The method of embodiment 62, wherein said methylation specific PCR comprises a MethyLight protocol. 
     Embodiment 65: The method of embodiment 62, wherein TaqMan PCR reactions are performed with primers specific for bisulfite-converted methylated and/or unmethylated sequences. 
     Embodiment 66: The method according to any one of embodiments 62-65, wherein said MSP utilizes one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences. 
     Embodiment 67: The method of embodiment 66, wherein said fluorescent probes comprise a fluorescent reporter dye and a quencher dye where the probe provides a signal upon cleavage by 5′ to 3′ nuclease activity of Taq DNA polymerase. 
     Embodiment 68: The method according to any one of embodiments 66-67, wherein a methylation signal is determined by the combined signal for a plurality of probes each specific to a different methylated region in an amplified region of interest. 
     Embodiment 69: The method according to any one of embodiments 66-67, wherein a methylation signal is determined by a plurality of probes specific for the same methylated region in an amplified region of interest. 
     Embodiment 70: The method according to any one of embodiments 66-67, wherein said plurality of probes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes. 
     Embodiment 71: The method according to any one of embodiments 66-67, wherein a methylation signal is determined by a single probe in the amplified region of interest. 
     Embodiment 72: The method according to any one of embodiments 66-71, wherein said probes are run in simplex or multiplex. 
     Embodiment 73: The method according to any one of embodiments 66-71, wherein said probes are run in a multiplex format. 
     Embodiment 74: The method according to any one of embodiments 66-73, wherein said probes are run as a nested PCR reaction. 
     Embodiment 75: The method according to any one of embodiments 66-74, wherein said PCR reaction comprises a bisulfite contamination control probe that that undergoes bisulfite-mediated cleavage during PCR if bisulfite is present in the reaction. 
     Embodiment 76: The method according to any one of embodiments 1-75, wherein PCR is performed for one or more mutated genes. 
     Embodiment 77: The method according to any one of embodiments 1-76, wherein PCR is performed for unconverted DNA as a control. 
     Embodiment 78: The method according to any one of embodiments 1-77, wherein PCR is performed for converted DNA as a control. 
     Embodiment 79: The method of embodiment 77, wherein PCR is performed for unconverted DNA where the unconverted DNA is a target for said method. 
     Embodiment 80: The method according to any one of embodiments 1-79, wherein a bisulfite reaction and a PCR reaction, or a desulphonation reaction and a PCR reaction, or a bisulfite reaction, a desulphonation reaction and a PCR reaction are all performed in the same reaction tube or chamber. 
     Embodiment 81: The method according to any one of embodiments 1-80, wherein said contacting a biological sample comprising a nucleic acid to a first matrix material comprises contacting a sample containing RNA to said first matrix material, where said matrix material binds said RNA thereby purifies the RNA. 
     Embodiment 82: The method of embodiment 81, wherein said method comprises eluting said RNA from said matrix material substantially independently of the DNA. 
     Embodiment 83: The method of embodiment 82, wherein the RNA is eluted from said first matrix material using a Tris buffered elution. 
     Embodiment 84: The method according to any one of embodiments 81-83, wherein said RNA is eluted and stored in a chamber. 
     Embodiment 85: The method according to any one of embodiments 81-84, wherein reverse transcription (RT) is performed on said RNA and qRT-PCR is performed to determine the level of target RNA sequences. 
     Embodiment 86: The method according to any one of embodiments 82-85, where the RNA fraction is used to elute the bisulfite converted nucleic acid from said second matrix material and mix with the bisulfite-converted DNA, or is mixed with eluted bisulfite-converted DNA. 
     Embodiment 87: The method of embodiment 86, wherein RT is performed on said RNA prior to, or after, combination with the bisulfite-converted DNA. 
     Embodiment 88: The method according to any one of embodiments 86-87, wherein qRT-PCR is performed for RT RNA in the mixture to determine the level of target RNA sequences and methylation specific PCR is performed on the mixture to determine methylation of target DNA sequences. 
     Embodiment 89: The method according to any one of embodiments 1-88, where methylation is determined for a promoter region of a gene selected from the group consisting of MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1. 
     Embodiment 90: The method according to any one of embodiments 81-89, wherein the expression level of RNA is determined for a methyltransferase. 
     Embodiment 91: The method of embodiment 90, wherein the expression level of RNA is determined for a methyltransferase selected from the group consisting of DNMT1, DNMT2, DNMT3A, DNMT3B, and TNMT3L. 
     Embodiment 92: A cartridge for determining the methylation state of a nucleic acid, said cartridge comprising: a column comprising a first matrix material, a sample receiving chamber, a temperature controlled channel or chamber, a plurality of chambers containing reagents and/or buffers, and when in use at least one of said chambers contains a bisulfite reagent, and at least one of said chambers contains a desulphonation/elution buffer, and wherein said cartridge optionally comprises a second column comprising said second matrix material. 
     Embodiment 93: The cartridge of embodiment 92, wherein said cartridge, when in use, comprises a chamber containing a reagent comprising guanidinium thiocyanate ethanol (GTC-EtOH). 
     Embodiment 94: The cartridge according to any one of embodiments 92-93, wherein said second column is absent. 
     Embodiment 95: The cartridge according to any one of embodiments 92-93, wherein said second column is present. 
     Embodiment 96: The cartridge according to any one of embodiments 92-95, wherein said temperature controlled channel or chamber is a thermocycling channel or chamber. 
     Embodiment 97: The cartridge according to any one of embodiments 92-96, wherein said cartridge further comprises a second heating channel or chamber. 
     Embodiment 98: The cartridge according to any one of embodiment 92-97, wherein said bisulfite reagent comprises a compound selected from the group consisting of ammonium bisulfite, sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO. 
     Embodiment 99: The cartridge of embodiment 98, wherein said bisulfite reagent comprises ammonium bisulfite. 
     Embodiment 100: The cartridge according to any one of embodiments 92-99, wherein said bisulfite is provided in a reagent mix comprising scavengers to prevent sulfite oxidation and/or catalysts. 
     Embodiment 101: The cartridge of embodiment 100, wherein said bisulfite is provided in a reagent mix comprising scavengers selected from the group consisting of TROLOX water-soluble analog of vitamin E and hydroquinone. 
     Embodiment 102: The cartridge according to any one of embodiments 100-101, wherein said bisulfite is provided in a reagent mix comprising polyamines as catalysts. 
     Embodiment 103: The cartridge according to any one of embodiments 92-102, wherein said first matrix material and/or said second matrix material, when present, comprises a material is selected from the group consisting of glass or silica, an ion exchange resin, and hydroxyapatite. 
     Embodiment 104: The cartridge according to any one of embodiments 92-103, wherein said cartridge comprises one or more chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer. 
     Embodiment 105: The cartridge of embodiment 104, wherein said cartridge contains at least two chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer. 
     Embodiment 106: The cartridge according to any one of embodiments 92-105, wherein said cartridge contains at least one chamber containing primers and probes for detection of methylation of a forward strand of a converted DNA. 
     Embodiment 107: The cartridge according to any one of embodiments 92-106, wherein said cartridge contains at least one chamber containing primers and probes for detection of methylation of a reverse strand of a converted DNA. 
     Embodiment 108: The cartridge according to any of embodiments 104-107, wherein said PCR primers, and/or probes, and/or enzymes are provided as beads. 
     Embodiment 109: The cartridge according to any one of embodiments 92-108, wherein said sample receiving chamber, said column(s), said plurality of chambers, and said temperature-controlled heating channel or chamber, are selectively in fluid communication. 
     Embodiment 110: The cartridge of embodiment 109, wherein said sample receiving chamber, said column(s), said plurality of chambers, and said temperature controlled channel or chamber, are selectively in fluid communication by microfluidic channels and valves. 
     Embodiment 111: The cartridge of embodiment 109, wherein said sample receiving chamber, said column(s), said plurality of chambers, and said temperature controlled channel or chamber or a port into said temperature controlled channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve. 
     Embodiment 112: The cartridge according to any one of embodiments 92-111, wherein said cartridge is configured so that, when in use, said cartridge comprises: 
     a first chamber containing a sample; 
     a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution; 
     a third chamber containing a bisulfite reagent; 
     a fourth chamber containing a buffer; 
     a fifth chamber containing a rinse solution; and 
     a sixth chamber containing an elution/desulphonation reagent. 
     Embodiment 113: The cartridge of embodiment 112, wherein said first chamber contains said sample in a GTC-EtOH-Tween extraction/precipitation reagent. 
     Embodiment 114: The cartridge according to any one of embodiments 92-113, wherein the cartridge is configured for the bisulfite reagent to be added to the cartridge at or near the time the sample is placed in the cartridge. 
     Embodiment 115: The cartridge according to any one of embodiments 92-113, wherein the bisulfite reagent is provided as a component of the cartridge. 
     Embodiment 116: The cartridge according to any one of embodiments 92-115, wherein the cartridge is configured for addition of GTC-ETOH-Tween buffer at or near the time the sample is placed into the cartridge. 
     Embodiment 117: The cartridge according to any one of embodiments 92-115, wherein the GTC-ETOH-Tween buffer is provided as a component of the cartridge. 
     Embodiment 118: The cartridge according to any one of embodiments 92-117, wherein said cartridge comprises a seventh chamber containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 119: The cartridge according to any one of embodiments 92-118, wherein said cartridge comprises an eighth chamber also containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 120: The cartridge according to any one of embodiments 92-119, wherein said cartridge comprises one or more chambers containing primers specific for bisulfite-converted methylated and/or unmethylated sequences. 
     Embodiment 121: The cartridge according to any one of embodiments 92-120, wherein said cartridge comprises one or more chambers containing reagents for TaqMan PCR reactions. 
     Embodiment 122: The cartridge according to any one of embodiments 92-121, wherein said cartridge comprises one or more chambers containing one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences. 
     Embodiment 123: The cartridge of embodiment 122, wherein said probes comprise a fluorescent reporter dye and a quencher dye, where the probes provides a signal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNA polymerase. 
     Embodiment 124: The cartridge according to any one of embodiments 122-123, wherein said cartridge comprises a plurality of probes each specific to a different methylated region in an amplified region of interest. 
     Embodiment 125: The cartridge according to any one of embodiments 122-123, wherein said cartridge comprises a single probe specific to a methylated region in an amplified region of interest. 
     Embodiment 126: The cartridge according to any one of embodiments 122-123, wherein said cartridge comprises a plurality of probes each specific to the same methylated region in an amplified region of interest. 
     Embodiment 127: The cartridge according to any one of embodiments 92-126, wherein said cartridge contains primers and/or probes to determine methylation of a promoter region of a gene selected from the group consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1. 
     Embodiment 128: The cartridge according to any one of embodiments 92-126, wherein said cartridge contains one or more primers shown in Tables 5, 9, or 10, and/or one or more probes shown in Tables 5, 9, or 10. 
     Embodiment 129: The cartridge of embodiment 128, wherein said cartridge contains the following probes and primers for determining methylation of MGMT using a nested PCR reaction: 
     an external forward primer (248b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
     
     an external reverse primer (249b) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
     
     an internal forward primer (250) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
     
     an internal reverse primer (251) comprising the nucleotide sequence 
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
 
     a probe (252a) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker. 
               
            
           
         
       
     
     Embodiment 130: The cartridge according to any one of embodiments 128-129, wherein said cartridge contains the following probes and primers for determining methylation of ACTB (e.g., as a control) using a nested PCR reaction: 
     an external forward primer (102) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
     
     an external reverse primer (103) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
     
     an internal forward primer (148) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 149) 
               
               
                   
                 GGTTTAGTAAGTTTTTTGGATTGTG; 
               
            
           
         
       
     
     an internal reverse primer (149) comprising the nucleotide sequence: 
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
 
     a probe (178) comprising the nucleotide sequence: fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 179) 
               
               
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker. 
               
            
           
         
       
     
     Embodiment 131: The cartridge according to any one of embodiments 92-130, wherein the cartridge is configured for determination of the expression level of RNA for a methyltransferase. 
     Embodiment 132: The cartridge of embodiment 131, wherein said methyltransferases is selected from the group consisting of DNMT1, DNMT2, DNMT3A, DNMT3B, and TNMT3L. 
     Embodiment 133: A system for determining the methylation of a nucleic acid in a biological sample, said system comprising: an enclosure configured to contain one or more sample processing modules, each sample processing module configured to hold a removable cartridge according to any one of embodiments 92-132; where said system is configured to operate the sample processing modules to perform sample processing to determine methylation of one or more target nucleic acids and optionally to determine the level of one or more target DNA sequences within a corresponding removable sample cartridge, wherein said processing on a sample within the corresponding removable sample cartridge performs a method according to any one of embodiments 1-91. 
     Embodiment 134: The system of embodiment 133, wherein said system is configured to contain one sample processing module. 
     Embodiment 135: The system of embodiment 133, wherein said system is configured to contain at least two sample processing modules, or at least 4 sample processing modules, or at least 8 sample processing modules, or at least 12 sample processing modules, or at least 16 sample processing modules, or at least 20 sample processing modules, or at least 24 sample processing modules, or at least 28 sample processing modules, or at least 32 sample processing modules, or at least 64 sample processing modules, or at least 128 sample processing modules. 
     Embodiment 136: The system according to any one of embodiments 133-135, wherein said modules comprise one or more heating plates to heat a temperature controlled chamber or channel in said cartridge. 
     Embodiment 137: The system according to any one of embodiments 133-136, wherein said modules comprise a fan configured to cool a temperature controlled channel or chamber in said cartridge. 
     Embodiment 138: The system according to any one of embodiments 133-137, wherein said modules comprise circuitry to pass information (e.g., optical information) to a computer for analysis. 
     Embodiment 139: The system according to any one of embodiments 133-138, wherein said modules comprise optical blocks to provide excitation and/or detection of one or more optical signals produced by reactions in said cartridge. 
     Embodiment 140: The system according to any one of embodiments 133-139, wherein said system is configured to operate said cartridge to perform a method according to any one of embodiments 1-91. 
     Embodiment 141: The system according to any one of embodiments 133-139, wherein said system is configured to operate said cartridge to: bind a sample to a column; elute DNA from the column and combine said DNA with a conversion reagent; heat the DNA/conversion reagent solution in a reaction chamber or tube to produce converted DNA; bind the converted DNA to a column; desulphonate and elute the DNA from the column; and perform PCR on the eluted desulphonated DNA in a reaction chamber or tube. 
     Embodiment 142: The system of embodiment 141, wherein said PCR is performed in the same reaction chamber or tube where the DNA/conversion reagent solution was previously heated. 
     Embodiment 143: A cartridge for sample preparation, said cartridge comprising: a channel or chamber comprising an affinity matrix that binds DNA, a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with said central valve assembly where said central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve wherein said plurality of chambers comprises: a chamber configured to receive up to about 5 ml or up to about 4 ml of sample solution; a chamber containing PEG; a chamber containing GTC-EtOH; a chamber containing an alkaline solution; and a chamber containing a buffer. 
     Embodiment 144: The cartridge of embodiment 143, wherein said plurality of chambers further comprises a chamber containing a bisulfate reagent. 
     Embodiment 145: The cartridge according to any one of embodiments 143-144, wherein said plurality of chambers comprises a chamber containing a GTC-ethanol wash solution. 
     Embodiment 146: The cartridge of embodiment 145, wherein said GTC-ethanol wash solution comprises 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, and 50% ethanol. 
     Embodiment 147: The cartridge according to any one of embodiments 143-146, wherein said PEG comprises PEG200. 
     Embodiment 148: The cartridge according to any one of embodiments 143-147, wherein said alkaline solution comprises KOH. 
     Embodiment 149: The cartridge according to any one of embodiments 143-148, wherein said buffer comprises Tris. 
     Embodiment 150: The cartridge according to any one of embodiments 143-149. wherein said plurality of chambers comprises a chamber containing beads comprising one or more PCR primers and/or probes. 
     Embodiment 151: The cartridge according to any one of embodiments 143-150, wherein said chamber containing PEG contains about 1 ml of PEG. 
     Embodiment 152. The cartridge according to any one of embodiments 143-151, wherein said chamber containing an alkaline solution contains about 500 μL of solution. 
     Embodiment 153: The cartridge according to any one of embodiments 143-152, wherein said chamber containing GTC-EtOH contains about 2 ml GTC-EtOH. 
     Embodiment 154: The cartridge according to any one of embodiments 143-153, wherein said chamber containing a buffer contains about 2 mL of buffer. 
     Embodiment 155: A high volume sample preparation (HVSP), said cartridge comprising: a channel or chamber comprising an affinity matrix that binds DNA, a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with said central valve assembly where said central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve wherein said plurality of chambers comprises: at least two different chambers each configured to receive up to about 4.5 ml of sample solution; a chamber containing PEG; a chamber containing an alkaline solution; and a chamber containing a buffer. 
     Embodiment 156: The cartridge of embodiment 155, wherein said plurality of chambers comprises at least three different chambers each configured to receive up to 4 ml of sample solution. 
     Embodiment 157: The cartridge according to any one of embodiments 155-156, wherein said PEG comprises PEG200. 
     Embodiment 158: The cartridge according to any one of embodiments 155-157, wherein said basic solution comprises KOH. 
     Embodiment 159: The cartridge according to any one of embodiments 155-158, wherein said buffer comprises Tris. 
     Embodiment 160: The cartridge according to any one of embodiments 155-159, wherein said plurality of chambers comprises a chamber containing a wash solution. 
     Embodiment 161: The cartridge of embodiment 160, wherein said wash solution comprise 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, and 50% ethanol. 
     Embodiment 162: The cartridge according to any one of embodiments 155-161, wherein said cartridge comprises a chamber configured for removal of a processed sample. 
     Embodiment 163: The cartridge according to any one of embodiments 155-162, wherein said sample chambers, when in use contain sample solution, GTC and isopropanol. 
     Embodiment 164: The cartridge of embodiment 163, wherein said sample chambers, when in use contain sample solution, GTC and isopropanol in substantially equal volumes. 
     Embodiment 165: The cartridge according to any one of embodiments 155-164 wherein said cartridge, when in use, comprises 4 ml of sample solution disposed in each of said chambers configured to receive a sample. 
     Embodiment 166: The cartridge according to any one of embodiments 155-165, wherein said cartridge provides DNA or RNA recovery that is substantially linear with respect to the sample volume between 0.5 ml and about 4 ml of sample. 
     Embodiment 167: The cartridge according to any one of embodiments 155-166, wherein said cartridge contains or is configured to receive a conversion reagent. 
     Embodiment 168: The cartridge of embodiment 167, wherein said cartridge, when in use, performs a bisulfate conversion of DNA. 
     Embodiment 169: A lysis solution for preparation of a DNA sample from serum or plasma, said lysis solution comprising: GTC, a buffer, a detergent, and optionally an anti-foaming agent. 
     Embodiment 170: The lysis solution of embodiment 169, wherein said lysis solution for serum or plasma comprises GTC, Tris pH 7.0, Tween 20, and antifoam SE15. 
     Embodiment 171: The lysis solution of embodiment 170, wherein said lysis solution for serum or plasma comprises about 4.5M GTC, about 45 mM Tris pH 7.0, about 1% Tween20, and about 0.01% Antifoam SE15. 
     Embodiment 172: A lysis solution for preparation of a DNA sample from an FFPE sample. 
     Embodiment 173: The lysis solution of embodiment 172, wherein said lysis solution for FFPE samples comprises a buffer, a detergent, NaCl, MgCl 2 , a chelating agent, antifoam SE15, and sodium azide. 
     Embodiment 174: The lysis solution of embodiment 173, wherein said lysis solution for FFPE samples comprises about 1% Tween20, about 400 mM NaCl, about 25 mM EDTA, about 10 mM MgCl 2 , about 50 mM HEPES pH 7.2, about 0.01% antifoam SE15, and about 0.01% sodium azide. 
     Embodiment 175: A kit for the determination of DNA methylation, said kit comprising: a container containing a cartridge for determining the methylation state of a nucleic acid according to any one of embodiments 92-136. 
     Embodiment 176: The kit of embodiment 175, wherein said kit further comprises a container containing a lysis solution. 
     Embodiment 177: The kit of embodiment 176, wherein said lysis solution is a lysis solution for serum or plasma according to any one of embodiments 169-171. 
     Embodiment 178: The kit of embodiment 176, wherein said lysis solution is a lysis solution for an FFPE sample according to any one of embodiments 172-174. 
     Embodiment 179: The kit according to any one of embodiments 175-178, wherein said kit comprises a container containing proteinase K. 
     Embodiment 180: The kit according to any one of embodiments 175-179, wherein said kit comprises a conversion reagent in said cartridge or in a container separate from the cartridge. 
     Embodiment 181: The kit of embodiment 180, wherein said kit comprises said conversion reagent in a container separate from the cartridge. 
     Embodiment 182: The kit of embodiment 180, wherein said kit comprises said conversion reagent is provided in a chamber of the cartridge. 
     Embodiment 183: The according to any one of embodiments 180-182, wherein said conversion reagent comprises a compound selected from the group consisting of sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, and DABSO. 
     Embodiment 184: The kit of embodiment 183, wherein said conversion reagent comprises ammonium bisulfite. 
     Embodiment 185: The kit according to any one of embodiments 175-184, wherein said kit comprises a container containing a sample processing reagent. 
     Embodiment 186: The kit of embodiment 185, wherein said sample processing reagent comprises guanidium thiocyanate. 
     Embodiment 187: The kit according to any one of embodiments 185-186, wherein said sample processing reagent comprise ethanol. 
     Embodiment 188: The kit according to any one of embodiments 175-187, wherein said kit comprises a container containing a cartridge for sample preparation according to any one of embodiments 155-166. 
     Embodiment 189: The kit according to any one of embodiments 175-188, wherein said kit contains instructional materials teaching the use of said cartridge for the determination of DNA methylation. 
     Embodiment 190: A cartridge for the detection of methylation markers of a cancer, said cartridge comprising: a plurality of chambers and a thermocycling channel or chamber, wherein said plurality of chambers and a port into said thermocycling channel or chamber are disposed around a central valve assembly and selectively in fluid communication with said central valve assembly where said central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber or port in fluid communication with said central valve wherein said plurality of chambers comprises: a sample receiving chamber; a chamber containing or configured to receive a bisulfite reagent; a chamber containing a wash solution; a chamber containing a Tris buffer; a chamber containing an alkaline solution comprising KOH; a chamber containing beads that provide a PCR master mix; and a chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for a cancer. 
     Embodiment 191: The cartridge of embodiment 190, wherein said plurality of chambers comprises a chamber disposed to receive waste solutions. 
     Embodiment 192: The cartridge according to any of embodiments 190-191, wherein said bisulfite reagent comprises a compound selected from the group consisting of sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, and DABSO. 
     Embodiment 193: The cartridge of embodiment 192, wherein said bisulfite reagent comprises ammonium bisulfite. 
     Embodiment 194: The cartridge according to any of embodiments 190-193, wherein said wash solution comprises 1.25M GTC, 25 mM Tris pH 7.0, and 50% ethanol. 
     Embodiment 195: The cartridge according to any of embodiments 190-194, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, and lung cancer. 
     Embodiment 196: The cartridge of embodiment 195, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes for a nested PCR reaction. 
     Embodiment 197: The cartridge of embodiment 196, wherein said nested PCR comprises a first PCR reaction specific for converted DNA and a second PCR reaction specific for methylated CpGs. 
     Embodiment 198: The cartridge according to any one of embodiments 190-197, wherein said chamber containing beads that provide PCR primers and probes chamber contains beads that provide PCR primers and probes to detect methylation of a forward strand of converted DNA. 
     Embodiment 199: The cartridge according to any one of embodiments 190-198, wherein said chamber containing beads that provide PCR primers and probes chamber contains beads that provide PCR primers and probes to detect methylation of a reverse strand of converted DNA. 
     Embodiment 200: The cartridge according to any of embodiments 190-199, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one or more genes selected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA 7, and MGMT. 
     Embodiment 201: The cartridge according to any of embodiments 190-200, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for pancreatic cancer. 
     Embodiment 202: The cartridge of embodiment 201, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of ADAMTS1, and/or BNC1. 
     Embodiment 203: The cartridge of embodiment 202, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of ADAMTS1. 
     Embodiment 204: The cartridge according to any one of embodiments 202-203, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of BNC1. 
     Embodiment 205: The cartridge of embodiment 202, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or probes for ADAMTS1 and/or BNC1 shown in Tables 5, or 10. 
     Embodiment 206: The cartridge according to any of embodiments 190-200, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for breast cancer. 
     Embodiment 207: The cartridge of embodiment 206, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one, two, three, four, five, or all genes selected from the group consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1. 
     Embodiment 208: The cartridge of embodiment 207, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of BRCA1. 
     Embodiment 209: The cartridge according to any one of embodiments 207-208, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of RASSF1A. 
     Embodiment 210: The cartridge according to any one of embodiments 207-209, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of AKR1B1. 
     Embodiment 211: The cartridge according to any one of embodiments 207-210, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HOXB4. 
     Embodiment 212: The cartridge according to any one of embodiments 207-211, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HIST1H3C. 
     Embodiment 213: The cartridge according to any one of embodiments 207-212, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of RASGRF2. 
     Embodiment 214: The cartridge according to any one of embodiments 207-213, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of TM6SF1. 
     Embodiment 215: The cartridge according to any one of embodiments 207-214, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or one or more PCR probes shown in Tables 5, or 9. 
     Embodiment 216: The cartridge of embodiment 206, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of BRCA1. 
     Embodiment 217: The cartridge according to any of embodiments 190-200, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for lung cancer. 
     Embodiment 218: The cartridge of embodiment 217, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one, two, three, or all genes selected from the group consisting of CDO1, SOX17, TAC1, and HOXA7. 
     Embodiment 219: The cartridge of embodiment 218, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of CDO1. 
     Embodiment 220: The cartridge according to any one of embodiments 218-219, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of SOX17. 
     Embodiment 221: The cartridge according to any one of embodiments 218-220, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of TAC1. 
     Embodiment 222: The cartridge according to any one of embodiments 218-221, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HOXA7. 
     Embodiment 223: The cartridge according to any of embodiments 190-200, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for brain cancer. 
     Embodiment 224: The cartridge of embodiment 223, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of MGMT. 
     Embodiment 225: The cartridge of embodiment 224, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or probes for MGMT shown in Tables 5, or 10. 
     Embodiment 226: The cartridge of embodiment 225, wherein said cartridge contains the following probes and primers for determining methylation of MGMT using a nested PCR reaction: 
     an external forward primer (248b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
     
     an external reverse primer (249b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
     
     an internal forward primer (250) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
     
     an internal reverse primer (251) comprising the nucleotide sequence 
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
 
     a probe (252a) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker. 
               
            
           
         
       
     
     Embodiment 227: The cartridge according to any one of embodiments 225-226, wherein said cartridge contains the following probes and primers for determining methylation of ACTB (e.g., as a control) using a nested PCR reaction: 
     an external forward primer (102) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
     
     an external reverse primer (103) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
     
     an internal forward primer (148) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 149) 
               
               
                   
                 GGTTTAGTAAGTTTTTTGGATTGTG; 
               
            
           
         
       
     
     an internal reverse primer (149) comprising the nucleotide sequence 
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
 
     a probe (178) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 179) 
               
               
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker. 
               
            
           
         
       
     
     Embodiment 228: A method of preparing a sample of cfDNA from serum or plasma, said method comprising: 
     combining a proteinase K treated sample of serum or plasma with a lysis solution according to any one of embodiments 169-171, and an alcohol to form a sample solution; 
     loading said sample solution into a sample receiving chamber in a cartridge according to any one of embodiments 143-154, or into a sample receiving chamber in a cartridge according to any one of embodiments 155-168; and 
     operating said cartridge to bind DNA in said sample to said affinity matrix and then to wash and release said DNA from said matrix. 
     Embodiment 229: The method of embodiment 228, wherein said combining a proteinase K treated sample of serum or plasma comprises combining said sample, lysis solution and alcohol in proportions corresponding to about 1.3 ml proteinase K treated serum or plasma, 2.2 mL lysis solution; and about 1.5 ml alcohol. 
     Embodiment 230: The method according to any one of embodiments 228-229, wherein said alcohol comprises isopropanol. 
     Embodiment 231: The method according to any one of embodiments 228-230, wherein said sample comprises serum. 
     Embodiment 232: The method according to any one of embodiments 228-231, wherein said sample comprises plasma. 
     Embodiment 233: The method according to any one of embodiments 228-232, wherein said sample comprises serum. 
     Embodiment 234: The method according to any one of embodiments 228-233, wherein operating said cartridge comprises introducing said cartridge into a sample processing module in a system according to any one of embodiments 133-139. 
     Embodiment 235: The method according to any one of embodiments 228-234, wherein said method further comprises operating said cartridge to convert said DNA for methylation detection. 
     Embodiment 236: The method according to any one of embodiments 228-235, wherein said method further comprises operating said cartridge to perform one or more PCR reactions using said DNA or converted DNA a template. 
     Embodiment 237: The method according to any one of embodiments 228-234, wherein said loading comprises loading said sample solution into one or more sample receiving chambers in a cartridge according to any one of embodiments 155-165. 
     Embodiment 238: The method of embodiment 237, wherein said method further comprises transferring the released DNA to a second cartridge for methylation detection and/or PCR. 
     Embodiment 239: The method of embodiment 238, wherein said second cartridge is a cartridge according to any one of embodiments 92-132. 
     Embodiment 240: The method according to any one of embodiments 238-239, wherein said method further comprises operating said second cartridge to convert said DNA for methylation detection. 
     Embodiment 241: The method according to any one of embodiments 238-240, wherein said method further comprises operating said second cartridge to perform one or more PCR reactions using said DNA or converted DNA as a template. 
     Embodiment 242: The method according to any one of embodiments 238-241, wherein said operating said second cartridge comprises introducing said second cartridge into a sample processing module in a system according to any one of embodiments 133-139. 
     Embodiment 243: A method of preparing a DNA from an FFPE sample, said method comprising: 
     combining a formalin-fixed paraffin embedded sample with a lysis solution according to any one of embodiments 172-174; 
     heating said lysis solution containing said sample; adding an alcohol to said sample to form a sample solution; loading said sample solution into a sample receiving chamber in a cartridge according to any one of embodiments 143-154, or into a sample receiving chamber in a cartridge according to any one of embodiments 155-168; and 
     operating said cartridge to bind DNA in said sample to said affinity matrix and then to wash and release said DNA from said matrix. 
     Embodiment 244: The method of embodiment 243, wherein said heating comprises adding proteinase K to said sample and heating said sample. 
     Embodiment 245: The method of embodiment 244, wherein said heating comprises adding about 50 μL proteinase K to about 1.2 mL of FFPE lysis solution containing a FFPE sample. 
     Embodiment 246: The method according to any one of embodiments 243-245, wherein said heating comprises heating said lysis solution to a temperature ranging from about 50° C. to about 60° C. 
     Embodiment 247: The method of embodiment 246, wherein said heating comprises heating said lysis solution to a temperature of about 56° C. 
     Embodiment 248: The method according to any one of embodiments 243-247, wherein said heating is for a period of time ranging up to about 4 hours, or up to about 5 hours, or up to about 6 hours. 
     Embodiment 249: The method of embodiment 248, wherein said heating is for about 4 hours. 
     Embodiment 250: The method according to any one of embodiments 243-249, wherein said alcohol comprises ethanol. 
     Embodiment 251: The method according to any one of embodiments 243-250, wherein said method comprises adding alcohol to said lysis solution in a volume ratio of about 1:1 lysis solution:alcohol. 
     Embodiment 252: The method according to any one of embodiments 243-251, wherein operating said cartridge comprises introducing said cartridge into a sample processing module in a system according to any one of embodiments 133-139. 
     Embodiment 253: The method according to any one of embodiments 243-252, wherein said method further comprises operating said cartridge to convert said DNA for methylation detection. 
     Embodiment 254: The method according to any one of embodiments 243-253, wherein said method further comprises operating said cartridge to perform one or more PCR reactions using said DNA or converted DNA as a template. 
     Embodiment 255: The method according to any one of embodiments 243-251, wherein said loading comprise loading said sample solution into one or more sample receiving chambers in a cartridge according to any one of embodiments 155-165. 
     Embodiment 256: The method of embodiment 255, wherein said method further comprises transferring the released DNA to a second cartridge for methylation detection and/or PCR. 
     Embodiment 257: The method of embodiment 256, wherein said second cartridge is a cartridge according to any one of embodiments 92-132. 
     Embodiment 258: The method according to any one of embodiments 256-257, wherein said method further comprises operating said second cartridge to convert said DNA for methylation detection. 
     Embodiment 259: The method according to any one of embodiments 256-258, wherein said method further comprises operating said second cartridge to perform one or more PCR reactions using said DNA or converted DNA as a template. 
     Embodiment 260: The method according to any one of embodiments 256-259, wherein said operating said second cartridge comprises introducing said second cartridge into a sample processing module in a system according to any one of embodiments 133-139. 
     Embodiment 261: A method of detecting a cancer, and/or staging a cancer, and/or detecting the predisposition to a cancer in a subject, said method comprising: 
     providing a biological sample from said subject, wherein said biological sample comprises a DNA; 
     utilizing a cartridge according to any one of claims  190 - 225  to detect methylation of one or more gene promoters in said DNA whose methylation state is a marker for a cancer, where an increase in methylation of said one or more gene promoters is indicative of the presence of a cancer or a predisposition to a cancer or a stage of a cancer or precancer. 
     Embodiment 262: The method of embodiment 261, wherein said subject is a human. 
     Embodiment 263: The method according to any one of embodiments 261-262, wherein said cancer is a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, a lung cancer, a B cell lymphoma, a bronchus cancer, a colorectal cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a brain or central nervous system cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma. 
     Embodiment 264: The method according to any one of embodiments 261-262, wherein said cancer is a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, a lung cancer. 
     Embodiment 265: The method according to any one of embodiments 261-264, wherein said sample comprise a sample from serum or plasma. 
     Embodiment 266: The method according to any one of embodiments 261-264, wherein said sample comprise an FFPE sample. 
     Embodiment 267: The method according to any one of embodiments 261-266, wherein said one or more gene promoters comprise the promoters of one or more genes selected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA7, and MGMT. 
     Embodiment 268: The method according to any one of embodiments 261-266, wherein said cancer is pancreatic cancer and said one or more gene promoters comprise the promoters of one, two, three, or four genes selected from the group consisting of ADAMTS1, and BNC1. 
     Embodiment 269: The method of embodiment 268, wherein said one or more gene promoters comprise the promoter of ADAMTS1. 
     Embodiment 270: The method according to any one of embodiments 268-269, wherein said one or more gene promoters comprise the promoter of BNC1. 
     Embodiment 271: The method according to any one of embodiments 261-266, wherein said cancer is breast cancer and said one or more gene promoters comprise the promoters of one, two, three, four, five, or all genes selected from the group consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1. 
     Embodiment 272: The method of embodiment 271, wherein said one or more gene promoters comprise the promoter of BRCA1. 
     Embodiment 273: The method according to any one of embodiments 271-272, wherein said one or more gene promoters comprise the promoter of RASSF1A. 
     Embodiment 274: The method according to any one of embodiments 271-273, wherein said one or more gene promoters comprise the promoter of AKR1B1. 
     Embodiment 275: The method according to any one of embodiments 271-274, wherein said one or more gene promoters comprise the promoter of HOXB4. 
     Embodiment 276: The method according to any one of embodiments 271-275, wherein said one or more gene promoters comprise the promoter of HIST1H3C. 
     Embodiment 277: The method according to any one of embodiments 271-276, wherein said one or more gene promoters comprise the promoter of RASGRF2. 
     Embodiment 278: The method according to any one of embodiments 271-277, wherein said one or more gene promoters comprise the promoter of TM6SF1. 
     Embodiment 279: The method according to any one of embodiments 261-266, wherein said cancer is breast cancer and said one or more gene promoters comprise the promoter of BRCA1. 
     Embodiment 280: The method according to any one of embodiments 261-266, wherein said cancer is lung cancer and said one or more gene promoters comprise the promoters of one, two, three, for all genes selected from the group consisting of CDO1, SOX17, TAC1, and HOXA7. 
     Embodiment 281: The method of embodiment 280, wherein said one or more gene promoters comprise the promoter of CDO1. 
     Embodiment 282: The method according to any one of embodiments 280-281, wherein said one or more gene promoters comprise the promoter of SOX17. 
     Embodiment 283: The method according to any one of embodiments 280-282, wherein said one or more gene promoters comprise the promoter of TAC1. 
     Embodiment 284: The method according to any one of embodiments 280-283, wherein said one or more gene promoters comprise the promoter of HOXA7. 
     Embodiment 285: The method according to any one of embodiments 261-266, wherein said cancer is brain cancer and said one or more gene promoters comprise the promoter of MGMT. 
     Embodiment 286: A method of converting cytosine residues in a DNA to uracil, while leaving 5-methylcytosine residues substantially unaffected, said method comprising: 
     contacting a sample comprising DNA with DABSO to convert said DNA; 
     desulphonating the converted DNA, to produce a DNA in which cytosine residues are converted to uracil, but 5-methylcytosine residues substantially unaffected. 
     Embodiment 287: The method of embodiment 286, wherein said contacting comprises contacting said DNA with DABSO at a concentration ranging from about 2 M up to about 5 M. 
     Embodiment 288: The method of embodiment 286, wherein said contacting comprises contacting said DNA with DAB SO at a concentration of about 2.5 M. 
     Embodiment 289: The method according to any one of embodiments 286-288, wherein said DABSO is dissolved in an alkaline aqueous solution. 
     Embodiment 290: The method of embodiment 289, wherein said DABSO is dissolved in a solution comprising KOH. 
     Embodiment 291: The method according to any one of embodiments 286-290, wherein said contacting comprises heating the DABSO/DNA solution to a temperature ranging from about 55° C. to about 90° C. 
     Embodiment 292: The method according to any one of embodiments 286-291, wherein said DABSO is reacted with the DNA for a period of time ranging from about 15 minutes up to about 90 minutes. 
     Embodiment 293: The method according to any one of embodiments 286-292, wherein said desulphonating comprises contacting said converted DNA with an alkaline reagent. 
     Embodiment 294: The method of embodiment 293, wherein said alkaline reagent comprises KOH. 
     Embodiment 295: The method according to any one of embodiments 286-294, wherein said conversion and/or desulphonation is performed on the DNA bound to a column. 
     Embodiment 296: The method according to any one of embodiments 286-294, wherein said conversion and/or desulphonation is performed on the DNA in solution. 
     Embodiment 297: A method of analyzing DNA methylation, said method comprising: 
     providing a DNA sample; 
     converting DNA in said sample according to the method of any one of embodiments 286-296; and 
     performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on the converted nucleic acid to determine the methylation of said nucleic acid. 
     Embodiment 298: The method of embodiment 297, wherein said providing a DNA sample comprises preparing a sample according to any one of embodiments 228-234 or according to any one of embodiments 243-252. 
     Embodiment 299: A kit for detection of methylation state of a DNA, said kit comprising: 
     a container containing a conversion reagent comprising DABSO; and 
     a container containing a desulphonation reagent. 
     Embodiment 300: The kit of embodiment 299, wherein said kit comprises a column comprising an affinity matrix. 
     Embodiment 301: The kit according to any one of embodiments 299-300, wherein said kit comprises a container containing a binding buffer. 
     Embodiment 302: The kit according to any one of embodiments 299-301, wherein said kit comprises a container containing an elution buffer. 
     Embodiment 303: The kit according to any one of embodiments 299-302, wherein said kit comprises a container containing a wash buffer. 
     Embodiment 304: The kit according to any one of embodiments 299-303, wherein said kit comprises a container containing a lysis solution according to any one of embodiments 169-171, and/or a container containing a lysis solution according to any one of embodiments 172-174. 
     Embodiment 305: The kit according to any one of embodiments 299-304, wherein said kit comprises a cartridge according to any one of embodiments 143-155 and/or a cartridge according to any one of embodiments 155-166. 
     Embodiment 306: The kit according to any one of embodiments 299-305, said kit comprising instructional materials teaching the use of said kit to convert a nuclei acid for determination of the methylation state of said nucleic acid. 
     Embodiment 307: A set of primers and probes for the determination of methylation of MGMT using a nested PCR reaction, said set comprising the following primers and probes: 
     an external forward primer comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
     
     an external reverse primer comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
     
     an internal forward primer comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
     
     an internal reverse primer comprising the nucleotide sequence 
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
 
     a probe comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker. 
               
            
           
         
       
     
     Embodiment 308: A set of primers and probes for the determination of methylation of ACTB (e.g., as a control) using a nested PCR reaction, said set comprising the following primers and probes: 
     an external forward primer (102) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
     
     an external reverse primer (103) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
     
     an internal forward primer (148) comprising the nucleotide sequence 
                            (SEQ ID NO: 149)           GGTTTAGTAAGTTTTTTGGATTGTG;            
and
 
     an internal reverse primer (149) comprising the nucleotide sequence 
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
 
     a probe (178) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 179) 
               
               
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker. 
               
            
           
         
       
     
     Embodiment 309: A set of primers and probes for the determination of methylation of MGMT using a nested PCR reaction with determination of the methylation of ACTB as a control, comprising the primers and probes of embodiment 307 and the primers and probes of embodiment 308. 
     Embodiment 310: A method of determining the methylation of MGMT using methylation specific PCR said method comprising: 
     providing a converted (e.g., bisulfite converted) DNA containing a promoter region of the MGMT gene; 
     performing methylation specific PCR for MGMT methylation using a nested PCR reaction comprising the following primers and probes:
         an external forward primer comprising the nucleotide sequence GTT       

     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 TT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
         
         
           
             an external reverse primer comprising the nucleotide sequence 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
         
         
           
             an internal forward primer comprising the nucleotide sequence 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
         
         
           
             an internal reverse primer comprising the nucleotide sequence 
           
         
       
    
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
         a probe comprising the nucleotide sequence fluor-       

                            (SEQ ID NO: 268)           CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker;            
and
 
     detecting and/or quantifying the PCR amplification product to provide determine methylation of said MGMT gene. 
     Embodiment 311: The method of embodiment 310, wherein said method further comprises: 
     providing a converted (e.g., bisulfite converted) DNA containing a promoter region of the ACTB gene (e.g., as a control); 
     performing methylation specific PCR for ACTB methylation using a nested PCR reaction comprising the following primers and probes:
         an external forward primer comprising the nucleotide sequence       

     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
         
         
           
             an external reverse primer comprising the nucleotide sequence 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
         
         
           
             an internal forward primer comprising the nucleotide sequence 
           
         
       
    
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 149) 
               
               
                   
                 GGTTTAGTAAGTTTTTTGGATTGTG; 
               
            
           
         
       
         
         
           
             an internal reverse primer comprising the nucleotide sequence 
           
         
       
    
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
         a probe comprising the nucleotide sequence fluor-       

                            (SEQ ID NO: 179)           CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker;            
and
 
     detecting and/or quantifying the PCR amplification product to provide determine methylation of said ACTB gene. 
     Embodiment 312: The method according to any one of embodiments 310-311, wherein said methylation specific PCR for MGMT methylation and said methylation specific PCR for ACTB methylation are performed in a single multiplex PCR reaction. 
     Embodiment 313: The method according to any one of embodiments 310-312, wherein said methylation specific PCR is performed using a cartridge according to any one of embodiments 92-132. 
     Embodiment 314: The method of embodiment 313, wherein: said providing a converted DNA containing a promoter region of the MGMT gene comprises introducing an unconverted DNA containing a promoter region of the MGMT gene into said cartridge and operating said cartridge to convert said DNA in said cartridge using a conversion reagent; and/or said providing a converted DNA containing a promoter region of the ACTB gene comprises introducing an unconverted DNA containing a promoter region of the ACTB gene into said cartridge and operating said cartridge to convert said DNA in said cartridge using a conversion reagent. 
     Embodiment 315: The method of embodiment 314, wherein said conversion reagent comprises a compound selected from the group consisting of ammonium bisulfite, sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO. 
     Embodiment 316: The method according to any one of embodiments 313-315, wherein said operating said cartridge comprises heating said DNA and said conversion reagent in a thermocycling channel or chamber that is later used to perform said nested PCR reaction. 
     Embodiment 317: A set of cartridges for determining the methylation state of a nucleic acid, said set of cartridges comprising: a first cartridge comprising: 
     a sample receiving chamber; 
     a column comprising a first matrix material; 
     a temperature controlled channel or chamber; 
     a sample removal chamber; and 
     a plurality of chambers containing reagents and/or buffers, wherein when in use at least one of said chambers contains a bisulfite reagent; and 
     a second cartridge comprising: 
     a sample receiving chamber; 
     a column comprising a second matrix material; 
     a temperature controlled channel or chamber; and 
     a plurality of chambers containing reagents and/or buffers, wherein when in use at least one of said chambers contains a desulphonation and/or elution reagent. 
     Embodiment 318: The set of cartridges of embodiment 317, wherein the temperature controlled channel or chamber in said first cartridge is a thermocycling channel or chamber. 
     Embodiment 319: The set of cartridges according to any one of embodiments 317-318, wherein the temperature controlled channel or chamber in said second cartridge is a thermocycling channel or chamber. 
     Embodiment 320: The set of cartridges according to any one of embodiments 317-319, wherein said bisulfite reagent comprises a compound selected from the group consisting of ammonium bisulfite, sodium metabisulfite, potassium bisulfite, cesium bisulfite, and DABSO. 
     Embodiment 321: The set of cartridges of embodiment 320, wherein said bisulfite reagent comprises ammonium bisulfite. 
     Embodiment 322: The set of cartridges according to any one of embodiments 317-321, wherein said bisulfite is provided in a reagent mix comprising scavengers to prevent sulfite oxidation and/or catalysts. 
     Embodiment 323: The set of cartridges of embodiment 322, wherein said bisulfite is provided in a reagent mix comprising scavengers selected from the group consisting of TROLOX water-soluble analog of vitamin E and hydroquinone. 
     Embodiment 324: The set of cartridges according to any one of embodiments 322-323, wherein said bisulfite is provided in a reagent mix comprising polyamines as catalysts. 
     Embodiment 325: The set of cartridges according to any one of embodiments 317-324, wherein said first cartridge is configured for the bisulfite reagent to be added to the cartridge at or near the time the sample is placed in the cartridge. 
     Embodiment 326: The set of cartridges according to any one of embodiments 317-325, wherein the bisulfite reagent is provided as a component in one of said plurality of chambers in said first the cartridge. 
     Embodiment 327: The set of cartridges according to any one of 317-326, wherein said first matrix material and/or said second matrix material, independently comprise a material is selected from the group consisting of glass or silica, an ion exchange resin, and hydroxyapatite. 
     Embodiment 328: The set of cartridges of embodiment 327, wherein said first matrix material comprises glass fibers. 
     Embodiment 329: The set of cartridges according to any one of 327-328, wherein said second matrix material comprises glass fibers. 
     Embodiment 330: The set of cartridges according to any one of embodiments 317-329, wherein said first cartridge comprises two sample receiving chambers. 
     Embodiment 331: The set of cartridges according to any one of 317-330, wherein at least one chamber comprising the plurality of chambers in said second cartridge contains PCR primers, and/or PCR probes, and/or a PCR master mix. 
     Embodiment 332: The set of cartridges according to any one of 317-331, wherein, when said cartridge is in use, a chamber comprising the plurality of chambers in said first cartridge contains GTC-EtOH in a buffer. 
     Embodiment 333: The set of cartridges according to any one of 317-332, wherein, when said cartridge is in use, a chamber comprising the plurality of chambers in said second cartridge contains GTC-EtOH in a buffer. 
     Embodiment 334: The set of cartridges according to any one of 332-333, wherein the first cartridge is configured for addition of GTC-ETOH in a buffer at or near the time the sample is placed into the cartridge. 
     Embodiment 335: The set of cartridges according to any one of 332-333, wherein the GTC-ETOH in a buffer is provided as a component in a chamber comprising the plurality of chambers of the first cartridge. 
     Embodiment 336: The set of cartridges according to any one of 332-335, wherein the second cartridge is configured for addition of GTC-ETOH in a buffer at or near the time the sample is placed into the cartridge. 
     Embodiment 337: The set of cartridges according to any one of 332-336, wherein the GTC-ETOH in a buffer is provided as a component in a chamber comprising the plurality of chambers of the second cartridge. 
     Embodiment 338: The set of cartridges according to any one of embodiments 317-337, wherein said second cartridge comprises one or more chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer. 
     Embodiment 339: The set of cartridges of embodiment 338, wherein said second cartridge contains at least two chambers containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s), and PCR reaction buffer. 
     Embodiment 340: The set of cartridges according to any one of embodiments 317-339, wherein said second cartridge contains at least one chamber containing primers and probes for detection of methylation of a forward strand of a converted DNA. 
     Embodiment 341: The set of cartridges according to any one of embodiments 317-340, wherein said second cartridge contains at least one chamber containing primers and probes for detection of methylation of a reverse strand of a converted DNA. 
     Embodiment 342: The set of cartridges according to any of embodiments 338-341, wherein said PCR primers, and/or probes, and/or enzymes are provided as beads. 
     Embodiment 343: The set of cartridges according to any one of embodiments 317-342, wherein: 
     in said first cartridge said sample receiving chamber, said column, said plurality of chambers, said sample removal chamber, and said temperature-controlled heating channel or chamber, are selectively in fluid communication; and/or 
     in said second cartridge said sample receiving chamber, said column, said plurality of chambers, and said temperature-controlled heating channel or chamber, are selectively in fluid communication. 
     Embodiment 344: The set of cartridges of embodiment 343, wherein 
     in said first cartridge, said sample receiving chamber, said column, said plurality of chambers, said sample removal chamber, and said temperature controlled channel or chamber, are selectively in fluid communication by microfluidic channels and valves; and/or 
     in said second cartridge, said sample receiving chamber, said column, said plurality of chambers, and said temperature controlled channel or chamber, are selectively in fluid communication by microfluidic channels and valves. 
     Embodiment 345: The set of cartridges of embodiment 343, wherein 
     in said first cartridge, said sample receiving chamber, said column, said plurality of chambers, said sample removal chamber, and said temperature controlled channel or chamber or a port into said temperature controlled channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve; and/or 
     in said second cartridge, said sample receiving chamber, said column, said plurality of chambers, and said temperature controlled channel or chamber or a port into said temperature controlled channel or chamber, are disposed around a central valve and selectively in fluid communication with a channel in said central valve, wherein said central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with said central valve. 
     Embodiment 346: The set of cartridges according to any one of embodiments 317-345, wherein said first cartridge is configured so that, when in use, said first cartridge comprises: 
     a first chamber containing a sample; 
     a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution; 
     a third chamber containing a bisulfate reagent; 
     a fourth chamber containing a Tris buffer; 
     a fifth chamber polyethyleneglycol (PEG); and 
     a sixth chamber containing KOH. 
     Embodiment 347: The set of cartridges of embodiment 346, wherein said first chamber in said first cartridge contains said sample in a GTC-EtOH-Tween extraction/precipitation reagent. 
     Embodiment 348: The set of cartridges according to any one of embodiments 346-347, wherein said a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution contains GTC-Tris (pH 7), ethanol. 
     Embodiment 349: The set of cartridges according to any one of embodiments 317-336, wherein said second cartridge is configured so that, when in use, said second cartridge comprises: 
     a first chamber containing a sample; 
     a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution; 
     a third chamber containing a Tris buffer; 
     a fourth chamber containing polyethylene glycol (PEG); 
     a fifth chamber containing KOH; and 
     a sixth chamber containing a PCR enzyme bead. 
     Embodiment 350: The set of cartridges of embodiment 349, wherein said second cartridge comprises a seventh chamber containing a Tris bead, and an enzyme bead. 
     Embodiment 351: The set of cartridges according to any one of embodiments 349-350, wherein said said a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution contains GTC-Tris (pH 7), ethanol. 
     Embodiment 352: The set of cartridges according to any one of embodiments 317-351, wherein said second cartridge comprises a chamber containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 353: The set of cartridges according to any one of embodiments 317-352, wherein said second cartridge comprises an eighth chamber also containing PCR primers and/or probes and/or PCR enzymes. 
     Embodiment 354: The set of cartridges according to any one of embodiments 317-353, wherein said second cartridge comprises one or more chambers containing primers specific for bisulfate-converted methylated and/or unmethylated sequences. 
     Embodiment 355: The set of cartridges according to any one of embodiments 317-354, wherein said second cartridge comprises one or more chambers containing reagents for TaqMan PCR reactions. 
     Embodiment 356: The set of cartridges according to any one of embodiments 317-355, wherein said second cartridge comprises one or more chambers containing one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences. 
     Embodiment 357: The set of cartridges of embodiment 356, wherein said probes comprise a fluorescent reporter dye and a quencher dye, where the probes provides a signal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNA polymerase. 
     Embodiment 358: The set of cartridges according to any one of embodiments 356-357, wherein said second cartridge comprises a plurality of probes each specific to a different methylated region in an amplified region of interest. 
     Embodiment 359: The set of cartridges according to any one of embodiments 356-357, wherein said second cartridge comprises a single probe specific to a methylated region in an amplified region of interest. 
     Embodiment 360: The set of cartridges according to any one of embodiments 356-357, wherein said second cartridge comprises a plurality of probes each specific to the same methylated region in an amplified region of interest. 
     Embodiment 361: The set of cartridges according to any one of embodiments 317-360, wherein said second cartridge contains primers and/or probes to determine methylation of a promoter region of a gene selected from the group consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1. 
     Embodiment 362: The set of cartridges according to any one of embodiments 317-360, wherein said second cartridge contains one or more primers shown in Tables 5, 9, or 10, and/or one or more probes shown in Tables 5, 9, or 10. 
     Embodiment 363: The set of cartridges of embodiment 362, wherein said second cartridge contains the following probes and primers for determining methylation of MGMT using a nested PCR reaction: 
     an external forward primer (248b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
     
     an external reverse primer (249b) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
     
     an internal forward primer (250) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
     
     an internal reverse primer (251) comprising the nucleotide sequence 
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
 
     a probe (252a) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker. 
               
            
           
         
       
     
     Embodiment 364: The set of cartridges according to any one of embodiments 362-363, wherein said second cartridge contains the following probes and primers for determining methylation of ACTB (e.g., as a control) using a nested PCR reaction: 
     an external forward primer (102) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
     
     an external reverse primer (103) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
     
     an internal forward primer (148) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 149) 
               
               
                   
                 GGTTTAGTAAGTTTTTTGGATTGTG; 
               
            
           
         
       
     
     an internal reverse primer (149) comprising the nucleotide sequence 
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
 
     a probe (178) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 179) 
               
               
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker. 
               
            
           
         
       
     
     Embodiment 365: The set of cartridges according to any one of embodiments 317-360, wherein said second cartridge comprises one or more chambers containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters. 
     Embodiment 366: The set of cartridges of embodiment 365, wherein said second cartridge comprises one or more chambers containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, and lung cancer. 
     Embodiment 367: The set of cartridges according to any of embodiments 365-366, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, and lung cancer. 
     Embodiment 368: The set of cartridges of embodiment 367, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes for a nested PCR reaction. 
     Embodiment 369: The set of cartridges of embodiment 368, wherein said nested PCR comprises a first PCR reaction specific for converted DNA and a second PCR reaction specific for methylated CpGs. 
     Embodiment 370: The set of cartridges according to any one of embodiments 349-369, wherein said chamber containing beads that provide PCR primers and probes chamber contains beads that provide PCR primers and probes to detect methylation of a forward strand of converted DNA. 
     Embodiment 371: The set of cartridges according to any one of embodiments 349-370, wherein said chamber containing beads that provide PCR primers and probes chamber contains beads that provide PCR primers and probes to detect methylation of a reverse strand of converted DNA. 
     Embodiment 372: The set of cartridges according to any of embodiments 349-371, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one or more genes selected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA7, and MGMT. 
     Embodiment 373: The set of cartridges according to any of embodiments 349-372, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for pancreatic cancer. 
     Embodiment 374: The set of cartridges of embodiment 373, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of ADAMTS1, and/or BNC1. 
     Embodiment 375: The set of cartridges of embodiment 374, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of ADAMTS1. 
     Embodiment 376: The set of cartridges according to any one of embodiments 374-375, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of BNC1. 
     Embodiment 377: The set of cartridges of embodiment 374, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or probes for ADAMTS1 and/or BNC1 shown in Tables 5, or 10. 
     Embodiment 378: The set of cartridges according to any of embodiments 349-372, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for breast cancer. 
     Embodiment 379: The set of cartridges of embodiment 378, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one, two, three, four, five, or all genes selected from the group consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1. 
     Embodiment 380: The set of cartridges of embodiment 379, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of BRCA1. 
     Embodiment 381: The set of cartridges according to any one of embodiments 379-380, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of RASSF1A. 
     Embodiment 382: The set of cartridges according to any one of embodiments 379-381, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of AKR1B1. 
     Embodiment 383: The set of cartridges according to any one of embodiments 379-382, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HOXB4. 
     Embodiment 384: The set of cartridges according to any one of embodiments 379-383, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HIST1H3C. 
     Embodiment 385: The set of cartridges according to any one of embodiments 379-384, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of RASGRF2. 
     Embodiment 386: The set of cartridges according to any one of embodiments 379-385, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of TM6SF1. 
     Embodiment 387: The set of cartridges according to any one of embodiments 379-386, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or one or more PCR probes shown in Tables 5, or 9. 
     Embodiment 388: The set of cartridges of embodiment 378, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of BRCA1. 
     Embodiment 389: The set of cartridges according to any of embodiments 349-372, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for lung cancer. 
     Embodiment 390: The set of cartridges of embodiment 389, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoters of one, two, three, or all genes selected from the group consisting of CDO1, SOX17, TAC1, and HOXA7. 
     Embodiment 391: The set of cartridges of embodiment 390, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of CDO1. 
     Embodiment 392: The set of cartridges according to any one of embodiments 390-391, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of SOX17. 
     Embodiment 393: The set of cartridges according to any one of embodiments 390-392, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of TAC1. 
     Embodiment 394: The set of cartridges according to any one of embodiments 390-393, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of HOXA7. 
     Embodiment 395: The set of cartridges according to any of embodiments 349-372, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of one or more gene promoters whose methylation state is a marker for brain cancer. 
     Embodiment 396: The set of cartridges of embodiment 395, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide PCR primers and probes to detect methylation of the promoter of MGMT. 
     Embodiment 397: The set of cartridges of embodiment 396, wherein said chamber containing beads that provide PCR primers and probes to detect methylation of one or more gene promoters comprises beads that provide one or more PCR primers and/or probes for MGMT shown in Tables 5, or 10. 
     Embodiment 398: The set of cartridges of embodiment 397, wherein said cartridge contains the following probes and primers for determining methylation of MGMT using a nested PCR reaction: 
     an external forward primer (248b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 263) 
               
               
                   
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT; 
               
            
           
         
       
     
     an external reverse primer (249b) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 AAAAAAC(T*)CCRCACTCTTCC; 
               
            
           
         
       
     
     an internal forward primer (250) comprising the nucleotide sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 TTTCGACGTTCGTAGGTTTTCGC; 
               
            
           
         
       
     
     an internal reverse primer (251) comprising the nucleotide sequence 
                            (SEQ ID NO: 267)           GCACTCTTCCGAAAACGAAACG;            
and
 
     a probe (252a) comprising the nucleotide sequence fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC-blocker. 
               
            
           
         
       
     
     Embodiment 399: The set of cartridges according to any one of embodiments 397-398, wherein said cartridge contains the following probes and primers for determining methylation of ACTB (e.g., as a control) using a nested PCR reaction: 
     an external forward primer (102) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 GTGATGGAGGAGGTTTAGTAAGTT; 
               
            
           
         
       
     
     an external reverse primer (103) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CCAATAAAACCTACTCCTCCCTTAA; 
               
            
           
         
       
     
     an internal forward primer (148) comprising the nucleotide sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 149) 
               
               
                   
                 GGTTTAGTAAGTTTTTTGGATTGTG; 
               
            
           
         
       
     
     an internal reverse primer (149) comprising the nucleotide sequence: 
                            (SEQ ID NO: 150)           CCTTAAAAATTACAAAAACCACAAC;            
and
 
     an probe (178) comprising the nucleotide sequence: 
     fluor- 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 179) 
               
               
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACAAACAC-blocker. 
               
            
           
         
       
     
     Embodiment 400: A system for determining the methylation of a nucleic acid in a biological sample, said system comprising: 
     an enclosure configured to contain one or more sample processing modules, each sample processing module configured to hold a removable cartridge first cartridge and/or second cartridge of said set of cartridges according to any one of embodiments 317-399; 
     where said system is configured to:
         operate the sample processing modules to perform sample processing to operate the first cartridge of said set of cartridges to perform a bisulfate conversion of a nucleic acid in a sample introduced into said first cartridge; and/or       

     to perform a desulphonation and to determine methylation of one or more target nucleic acids within a corresponding removable sample cartridge. 
     Embodiment 401: The system of embodiment 400, wherein said system is configured to contain one sample processing module. 
     Embodiment 402: The system of embodiment 400, wherein said system is configured to contain at least two sample processing modules, or at least 4 sample processing modules, or at least 8 sample processing modules, or at least 12 sample processing modules, or at least 16 sample processing modules, or at least 20 sample processing modules, or at least 24 sample processing modules, or at least 28 sample processing modules, or at least 32 sample processing modules, or at least 64 sample processing modules, or at least 128 sample processing modules. 
     Embodiment 403: The system according to any one of embodiments 400-402, wherein said modules comprise one or more heating plates to heat a temperature controlled chamber or channel in said cartridge. 
     Embodiment 404: The system according to any one of embodiments 400-403, wherein said modules comprise a fan configured to cool a temperature controlled channel or chamber in said cartridge. 
     Embodiment 405: The system according to any one of embodiments 400-404, wherein said modules comprise circuitry to pass information (e.g., optical information) to a computer for analysis. 
     Embodiment 406: The system according to any one of embodiments 400-405, wherein said modules comprise optical blocks to provide excitation and/or detection of one or more optical signals produced by reactions in said cartridge. 
     Embodiment 407: The system according to any one of embodiments 400-406, wherein said system is configured to operate said first cartridge of said set of cartridges to: 
     bind a sample to a column; 
     elute DNA from the column and combine said DNA with a conversion reagent; 
     heat the DNA/conversion reagent solution in a reaction chamber or tube to produce converted DNA; and 
     to deliver the converted DNA to a sample removal chamber in the first cartridge. 
     Embodiment 408: The system according to any one of embodiments 400-407, wherein said system is configured to operate said second cartridge in said set of cartridges to: 
     bind the converted DNA to a column; 
     wash, rinse, and elute the converted DNA; 
     elute the DNA from the column; and 
     defulphonate the converted DNA. 
     Embodiment 409: The system of embodiment 408, wherein said system is configured to operate said second cartridge in said set of cartridges to perform PCR on the eluted desulphonated DNA in a reaction chamber or tube. 
     Embodiment 410: A method of determining the methylation state of a nucleic acid, said method comprising: providing a biological sample in a sample chamber of a first cartridge in a set of cartridges according to any one of embodiments 317-399; and operating said first cartridge to: 
     bind DNA in said sample to said first matrix material; 
     wash the bound DNA; elute the bound DNA off of the matrix material; combine the eluted DNA with said bisulfite reagent; 
     heat the mixture of DNA and bisulfite reagent in said temperature controlled channel or chamber perform a bisulfite conversion of said DNA; and 
     deliver the bisulfite-converted DNA into the sample removal chamber of said first cartridge. 
     Embodiment 411: The method of embodiment 410, wherein said method further comprise: 
     providing bisulfite converted DNA in a sample chamber of a second cartridge in a set of cartridges according to any one of embodiments 317-399; and 
     operating said second cartridge to:
         bind said bisulfite converted DNA to said second matrix material;   wash the bound bisulfite-converted DNA;   elute the washed bisulfite-converted DNA from said second matrix material; and   desulphonate the bisulfite converted DNA.       

     Embodiment 412: The method of embodiment 411, wherein said second cartridge is operated to elute the bisulfite-converted DNA from said second matrix material before desulphonation. 
     Embodiment 413: The method of embodiment 411, wherein said second cartridge is operated to elute the bisulfite-converted DNA from said second matrix material after or during desulphonation. 
     Embodiment 414: The method according to any one of embodiments 411-413, wherein said method comprises operating said second cartridge to perform methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on said converted nucleic acid to determine the methylation of said nucleic acid. 
     Embodiment 415: The method according to any one of embodiments 410-414, wherein said sample comprises one or more samples selected from the group consisting of a cell, a tissue, and a biological fluid containing a nucleic acid. 
     Embodiment 416: The method of embodiment 415, wherein said biological sample comprises a biological fluid selected from the group consisting of whole blood, plasma, serum, saliva, mucus, urine, sputum, pancreatic juice, and cerebrospinal fluid. 
     Embodiment 417: The method of embodiment 415, wherein said biological sample comprises a sample selected from the group consisting of a tissue sample, a formalin fixed paraffin embedded (FFPE) tissue, fresh frozen tissue, fine needle aspirates (FNA), and a core biopsy. 
     Embodiment 418: The method according to any one of embodiments 410-417, wherein said method comprises contacting said biological sample with a lysis solution. 
     Embodiment 419: The method according to any one of embodiments 410-418, wherein operating said first cartridge comprises introducing said first cartridge into a sample processing module in a system according to any one of embodiments 400-409. 
     Embodiment 420: The method according to any one of embodiments 410-419, wherein operating said second cartridge comprises introducing said second cartridge into a sample processing module in a system according to any one of embodiments 400-409. 
     Embodiment 421: The method according to any one of embodiments 410-420, wherein said providing a biological sample in a sample chamber of a first cartridge comprises loading a sample into one or more sample receiving chambers in said first cartridge. 
     Embodiment 422: The method according to any one of embodiments 410-421, wherein said providing bisulfite converted DNA in a sample chamber of a second cartridge comprises transferring bisulfite-converted DNA from a sample removal chamber in said first cartridge into a sample receiving chamber of said second cartridge. 
     Embodiment 423: The method according to any one of embodiments 410-422, wherein said eluting the bound DNA in said first cartridge comprises eluting and denaturing said DNA using a low concentration of potassium hydroxide or other base. 
     Embodiment 424: The method of embodiment 423, wherein said eluting the bound DNA in said first cartridge comprises eluting and denaturing said DNA with an alkaline solution with a pH greater than about pH 10.5. 
     Embodiment 425: The method of embodiment 423, wherein said eluting the bound DNA in said first cartridge comprises eluting and denaturing said DNA with an alkaline solution with a pH greater than about pH 12. 
     Embodiment 426: The method of embodiments 424-425, wherein said alkaline solution is a 10-15 mM KOH solution. 
     Embodiment 427: The method according to any one of embodiments 410-426, wherein said combining the eluted DNA with a bisulfite reagent in said first cartridge comprises incubating the DNA in an ammonium bisulfite solution having a concentration that ranges from about 6M to about 7M. 
     Embodiment 428: The method of embodiment 427, wherein said combining the eluted DNA with a bisulfite reagent in said first cartridge comprises incubating the DNA in an ammonium bisulfite solution having a concentration of about 6.5M. 
     Embodiment 429: The method of embodiment 428, wherein said combining the eluted DNA with a bisulfite reagent in said first cartridge comprises transferring the DNA in a concentrated bisulfite solution into a temperature controlled channel or chamber in said first cartridge and heating said mixture. 
     Embodiment 430: The method of embodiment 429, wherein said incubating comprises thermally cycling the concentrated bisulfite solution from a temperature of about 60° C. to about 95° C. 
     Embodiment 431: The method according to any one of embodiments 411-430, wherein said binding the bisulfite converted DNA to the second matrix material comprises mixing the DNA-bisulfite solution with fresh GTC-EtOH in said second column, and dispensing the solution over said second matrix material. 
     Embodiment 432: The method of embodiment 431, wherein said method comprises washing the DNA in said second matrix material with fresh GTC-EtOH, and then a rinse solution. 
     Embodiment 433: The method of embodiment 432, wherein said rinse solution comprises PEG200. 
     Embodiment 434: The method according to any one of embodiments 411-433, wherein said desulphonating the converted DNA comprises eluting the DNA from said second matrix material with a high pH desulphonation buffer and incubating said solution. 
     Embodiment 435: The method of embodiment 434, wherein said incubating is for a period of time ranging from about 1 minute to about 1 hour, or from about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes, or for about 15 minutes. 
     Embodiment 436: The method of embodiments 434-435, wherein said high pH desulphonation/elution buffer comprises KOH. 
     Embodiment 437: The method according to any one of embodiments 411-436, wherein said method comprises operating said second cartridge to perform one or more PCR reactions using said converted DNA as a template. 
     Embodiment 438: The method of embodiment 437, wherein methylation specific PCR is performed to determine methylation of target nucleic acid sequences. 
     Embodiment 439: The method of embodiment 438, wherein said methylation specific PCR (MSP) is performed using primers specific for methylated sequences and/or primers specific for unmethylated sequences. 
     Embodiment 440: The method of embodiment 438, wherein said methylation specific PCR comprises a MethyLight protocol. 
     Embodiment 441: The method of embodiment 438, wherein TaqMan PCR reactions are performed with primers specific for bisulfate-converted methylated and/or unmethylated sequences. 
     Embodiment 442: The method according to any one of embodiments 438-441, wherein said methylation specific PCR (MSP) utilizes one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences. 
     Embodiment 443: The method of embodiment 442, wherein said fluorescent probes comprise a fluorescent reporter dye and a quencher dye where the probe provides a signal upon cleavage by 5′ to 3′ nuclease activity of Taq DNA polymerase. 
     Embodiment 444: The method according to any one of embodiments 442-443, wherein a methylation signal is determined by the combined signal for a plurality of probes each specific to a different methylated region in an amplified region of interest. 
     Embodiment 445: The method according to any one of embodiments 442-443, wherein a methylation signal is determined by a plurality of probes specific for the same methylated region in an amplified region of interest. 
     Embodiment 446: The method according to any one of embodiments 442-443, wherein said plurality of probes comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more probes. 
     Embodiment 447: The method according to any one of embodiments 442-443, wherein a methylation signal is determined by a single probe in the amplified region of interest. 
     Embodiment 448: The method according to any one of embodiments 442-447, wherein said probes are run in simplex or multiplex. 
     Embodiment 449: The method according to any one of embodiments 442-447, wherein said probes are run in a multiplex format. 
     Embodiment 450: The method according to any one of embodiments 442-449, wherein said probes are run as a nested PCR reaction. 
     Embodiment 451: The method according to any one of embodiments 442-450, wherein said PCR reaction comprises a bisulfite contamination control probe that that undergoes bisulfite-mediated cleavage during PCR if bisulfite is present in the reaction. 
     Embodiment 452: The method according to any one of embodiments 411-451, wherein PCR is performed for one or more mutated genes. 
     Embodiment 453: The method according to any one of embodiments 411-452, wherein PCR is performed for unconverted DNA as a control. 
     Embodiment 454: The method according to any one of embodiments 411-453, wherein PCR is performed for converted DNA as a control. 
     Embodiment 455: The method of embodiment 453, wherein PCR is performed for unconverted DNA where the unconverted DNA is a target for said method. 
     Embodiment 456: The method according to any one of embodiments 410-455, where methylation is determined for a promoter region of a gene selected from the group consisting of MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1. 
     Embodiment 457: A method of detecting a cancer or the predisposition to a cancer in a subject, said method comprising: 
     providing a biological sample from said subject, wherein said biological sample comprises a DNA; 
     utilizing a set of cartridges according to any one of embodiments 317-399, wherein said first cartridge of said set of cartridges is used to perform a bisulfite conversion of said DNA; and said second cartridge of said set of cartridges is used to desulphonate the converted DNA and to detect methylation of one or more gene promoters in said DNA whose methylation state is a marker for a cancer, where an increase in methylation of said one or more gene promoters is indicative of the presence of a cancer or a predisposition to a cancer or a stage of a cancer or precancer. 
     Embodiment 458: The method of embodiment 457, wherein said subject is a human. 
     Embodiment 459: The method according to any one of embodiments 457-458, wherein said cancer is a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, a lung cancer, a B cell lymphoma, a bronchus cancer, a colorectal cancer, a stomach cancer, an ovarian cancer, a urinary bladder cancer, a brain or central nervous system cancer, a peripheral nervous system cancer, an esophageal cancer, a cervical cancer, a melanoma, a uterine or endometrial cancer, a cancer of the oral cavity or pharynx, a liver cancer, a kidney cancer, a biliary tract cancer, a small bowel or appendix cancer, a salivary gland cancer, a thyroid gland cancer, a adrenal gland cancer, an osteosarcoma, a chondrosarcoma, a liposarcoma, a testes cancer, and a malignant fibrous histiocytoma. 
     Embodiment 460: The method according to any one of embodiments 457-458, wherein said cancer is a cancer selected from the group consisting of breast cancer, pancreatic cancer, prostate cancer, brain cancer, a lung cancer. 
     Embodiment 461: The method according to any one of embodiments 457-460, wherein said sample comprise a sample from serum or plasma. 
     Embodiment 462: The method according to any one of embodiments 457-460, wherein said sample comprise an FFPE sample. 
     Embodiment 463: The method according to any one of embodiments 457-462, wherein said one or more gene promoters comprise the promoters of one or more genes selected from the group consisting of RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, BRCA1, BNC1, ADAMTS1, CDO1, SOX17, TAC1, HOXA 7, and MGMT. 
     Embodiment 464: The method according to any one of embodiments 457-462, wherein said cancer is pancreatic cancer and said one or more gene promoters comprise the promoters of one, two, three, or four genes selected from the group consisting of ADAMTS1, and BNC1. 
     Embodiment 465: The method of embodiment 464, wherein said one or more gene promoters comprise the promoter of ADAMTS1. 
     Embodiment 466: The method according to any one of embodiments 464-465, wherein said one or more gene promoters comprise the promoter of BNC1. 
     Embodiment 467: The method according to any one of embodiments 457-462, wherein said cancer is breast cancer and said one or more gene promoters comprise the promoters of one, two, three, four, five, or all genes selected from the group consisting of BRCA1, RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, and TM6SF1. 
     Embodiment 468: The method of embodiment 467, wherein said one or more gene promoters comprise the promoter of BRCA1. 
     Embodiment 469: The method according to any one of embodiments 467-468, wherein said one or more gene promoters comprise the promoter of RASSF1A. 
     Embodiment 470: The method according to any one of embodiments 467-469, wherein said one or more gene promoters comprise the promoter of AKR1B1. 
     Embodiment 471: The method according to any one of embodiments 467-470, wherein said one or more gene promoters comprise the promoter of HOXB4. 
     Embodiment 472: The method according to any one of embodiments 467-471, wherein said one or more gene promoters comprise the promoter of HIST1H3C. 
     Embodiment 473: The method according to any one of embodiments 467-472, wherein said one or more gene promoters comprise the promoter of RASGRF2. 
     Embodiment 474: The method according to any one of embodiments 467-473, wherein said one or more gene promoters comprise the promoter of TM6SF1. 
     Embodiment 475: The method according to any one of embodiments 457-462, wherein said cancer is breast cancer and said one or more gene promoters comprise the promoter of BRCA1. 
     Embodiment 476: The method according to any one of embodiments 457-462, wherein said cancer is lung cancer and said one or more gene promoters comprise the promoters of one, two, three, for all genes selected from the group consisting of CDO1, SOX17, TAC1, and HOXA7. 
     Embodiment 477: The method of embodiment 476, wherein said one or more gene promoters comprise the promoter of CDO1. 
     Embodiment 478: The method according to any one of embodiments 476-477, wherein said one or more gene promoters comprise the promoter of SOX17. 
     Embodiment 479: The method according to any one of embodiments 476-478, wherein said one or more gene promoters comprise the promoter of TAC1. 
     Embodiment 480: The method according to any one of embodiments 476-479, wherein said one or more gene promoters comprise the promoter of HOXA7. 
     Embodiment 481: The method according to any one of embodiments 457-462, wherein said cancer is brain cancer and said one or more gene promoters comprise the promoter of MGMT. 
     Embodiment 482: A kit for the determination of DNA methylation, said kit comprising: a container containing a first cartridge and/or a second cartridge of set of cartridges according to any one of embodiments 317-399. 
     Embodiment 483: The kit of embodiment 482, wherein said first cartridge and said second cartridge are contained in the same container. 
     Embodiment 484: The kit of embodiment 482, wherein said first cartridge and said second cartridge are in separate containers. 
     Embodiment 485: The kit of embodiment 482, wherein said kit further comprises a container containing a lysis solution. 
     Embodiment 486: The kit of embodiment 485, wherein said lysis solution is a lysis solution for serum or plasma. 
     Embodiment 487: The kit of embodiments 485, wherein said lysis solution is a lysis solution for an FFPE sample. 
     Embodiment 488: The kit according to any one of embodiments 482-487, wherein said kit comprises a container containing proteinase K. 
     Embodiment 489: The kit according to any one of embodiments 482-488, wherein said kit comprises a conversion reagent in said cartridge or in a container separate from the cartridge. 
     Embodiment 490: The kit of embodiment 489, wherein said kit comprises said conversion reagent in a container separate from the cartridge. 
     Embodiment 491: The kit of embodiment 489, wherein said kit comprises said conversion reagent is provided in a chamber of the cartridge. 
     Embodiment 492: The according to any one of embodiments 489-491, wherein said conversion reagent comprises a compound selected from the group consisting of sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, and DABSO. 
     Embodiment 493: The kit of embodiment 492, wherein said conversion reagent comprises ammonium bisulfite. 
     Embodiment 494: The kit according to any one of embodiments 482-493, wherein said kit comprises a container containing a sample processing reagent. 
     Embodiment 495: The kit of embodiment 494, wherein said sample processing reagent comprises guanidium thiocyanate. 
     Embodiment 496: The kit according to any one of embodiments 494-495, wherein said sample processing reagent comprise ethanol. 
     Embodiment 497: The kit according to any one of embodiments 482-496, wherein said kit contains instructional materials teaching the use of said cartridge for the determination of DNA methylation. 
     In certain embodiments the methods and/or cartridges expressly exclude magnetic materials including magnetic glass, magnetic hydroxyapatite, and magnetic matrix materials. In certain embodiments the methods and/or cartridges expressly exclude magnetic materials for DNA isolation. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  illustrates major components of a cartridge (e.g., a GENEXPERT® cartridge) suitable for use with the methods described herein.  FIG. 1B  shows a top view of the cartridge illustrating chambers disposed around a central valve.  FIG. 1C  shows an illustrative workflow for the determination of DNA methylation utilizing the reaction cartridge. 
         FIG. 2 , panels A-C, illustrates one embodiment of a GENEXPERT® cartridge suitable for the determination of DNA methylation as described herein. 
         FIGS. 3A-3C  show illustrative, but non-limiting embodiments of the modules, and systems (e.g., processing units) for the determination of DNA methylation.  FIG. 3A  illustrates a module for operation of a GENEXPERT® cartridge.  FIG. 3B  illustrates some components of one embodiment of a module for operation of a cartridge for the analysis of DNA methylation.  FIG. 3C  illustrates a system (e.g., processing unit) incorporating a plurality of modules. 
         FIGS. 4A-4D  illustrate various strategies for the use of MethyLight protocols to detect/quantify DNA phosphorylation.  FIG. 4A , modified from Eads et al. 92000)  Nucleic Acids. Res.,  28(8): e32) schematically illustrates MethyLight technology. DNA is modified by sodium bisulfate which generates methylation-dependent sequence differences, e.g., at CpG dinucleotides by converting unmethylated cytosine residues (locations indicated by white circles) to uracil, while methylated cytosine residues (locations indicated by black circles) are retained as cytosine. Fluorescence-based PCR is then performed with primers that either overlap methylation sites or that do not overlap any methylation sites. Sequence discrimination can occur either at the level of the PCR amplification process (panel D) or at the level of the probe hybridization process (panel B), or both (panel D). Sequence discrimination at the PCR amplification level utilizes primers and probes (panel D), or just primers (panel C), to overlap potential methylation sites (e.g., CpG dinucleotides). Only two (fully methylated (M) and fully unmethylated (U)) of the many theoretical methylation permutations are shown. The MethyLight assay can also be designed such that sequence discrimination does not occur at the PCR amplification level. If neither the primers nor the probe overlap sites of methylation (e.g., CpG dinucleotides) (panel A), then no methylation-dependent sequence discrimination occurs at the PCR amplification or probe hybridization level. This reaction represents amplification of the converted genomic DNA without bias to methylation status, which can serve as a control for the amount of input DNA. When just the probe overlaps methylation sites (panel B), then sequence discrimination can occur through probe hybridization.  FIG. 4B  illustrates a MethyLight approach using a single, e.g., methylation-specific, probe (PR3) along with methylation specific forward (FW) and reverse (RV) primers.  FIG. 4C  illustrates a MethyLight approach using multiple probes (PR1 . . . PR5) that each target different regions.  FIG. 4D  illustrates a MethyLight approach using multiple probes (PR1 . . . PR5) that each target the same region, but provide signals for different methylation patterns. 
         FIG. 5  illustrates results from a representative GeneXpert run from 300 ng of HGDNA showing an ACTB qPCR curve and an HMBS qPCR curve. 
         FIGS. 6A and 6B  illustrate the results of a titration for bisulfate-converted ACTB using human genomic DNA (hgDNA) in a 15 cycle nested qPCR ( FIG. 6A ) and a 20 cycle nested qPCR ( FIG. 6B ). 
         FIGS. 7A-7C  shows the result of 20 cycles of nested qPCR (in the cartridge) for six methylated targets (AKR1B1, HOXB4, TM6SF1, RAASGRF2, and RASSF1A).  FIG. 7A  shows the results for 25 ng of HSDNA or 5000 cells without bisulfite conversion.  FIG. 7B  shows the results of 20 cycles of nested qPCR for the bisulfite converted methylated targets using DNA from MBA-453 cells.  FIG. 7C  shows the results of 20 cycles of nested qPCR for the bisulfite converted methylated targets using DNA from MBA-453 cells in a carrier (1 μg of SS and 10 ng of HS DNA). Fallouts occur at around 25-50 copies or around 100 cells. 
         FIG. 8  illustrates the results of a determination of conversion efficiency. The conversion efficiency is about 66% (˜1 Ct) the difference between unconverted HMBS and converted ACTB. 
         FIG. 9  illustrates the increase in specificity for converted DNA produced by nested qPCR. 
         FIG. 10  illustrates the specificity of the methylation cartridge. There is no priming off of unconverted DNA (top panel) or unmethylated DNA (bottom panel) except for HIST1H3C. 
         FIG. 11  shows illustrative but non-limiting workflows for analysis of methylation using a cartridge (e.g., a GENEXPERT® cartridge). Top illustrates one work flow for analysis of DNA methylation in a serum or plasma sample. Bottom illustrates one work flow for analysis of DNA methylation in a tissue section (e.g., frozen or formalin-fixed paraffin embedded (FFPE) section). 
         FIG. 12  illustrates the results for a FFPE cell button for converted ALU (left-most curve) and methylated RASSF1A (right-most curve). 
         FIG. 13A  illustrates a cartridge layout and  FIG. 13B  illustrates a flow chart of the protocol used in Example 4. 
         FIG. 14  illustrates a run in which some samples contain bisulfite contamination. 
         FIG. 15A  illustrates the results of 1000 MBA-453 cells with bisulfite conversion (with HOXB4 giving the greatest signal).  FIG. 15B  illustrates results of 25 ng of HS DNA control (with only HIST1H3C showing a detectable signal). 
         FIG. 16  illustrates the structure of DABSO (1,4-diazoniabicyclo[2.2.2]octane-1,4-disulfinate). 
         FIG. 17  illustrates one embodiment of a cfDNA sample preparation cartridge. The cartridge is effective for both DNA and RNA isolation. The cartridge provides three GTC-ethanol washes (GTC-ethanol washes are typically 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, 50% ethanol), a PEG200 rinse, and a 15 mM KOH elution. 
         FIG. 18  illustrates controls for cfDNA extraction. 
         FIG. 19A  shows a comparison of cfDNA preparation using a sample preparation cartridge as described herein compared to a standard tube-fill (i.e. tube-based kit) preparation (left-most curve). The cartridge preparation yield is very comparable to that obtained using a tube fill method (right-most curve).  FIG. 19B  shows a comparison of the amount of extracted DNA detected using a cartridge-based DNA cleanup as compared to a standard tube-fill as a function of DNA amount. The cartridge-based method is conservatively within 1 Ct of the tube-fill methods and is believed to be closer at higher DNA concentration. 
         FIG. 20A  illustrates one embodiment of a high-volume (e.g., up to 12 ml) sample preparation (HVSP) cartridge that can be used with a qPCR cartridge and/or with a methylation detection cartridge.  FIG. 20B  schematically illustrates one variation of work flows in the HVSP cartridge when used in combination with a qPCR cartridge to perform a methylation analysis. 
         FIG. 21  illustrates the detection of HBMS or β-globin using a two cartridge cleanup using a high-volume sample preparation cartridge (see, e.g.,  FIG. 20 ) where the sample is transferred from the high volume cartridge to the PCR analysis cartridge compared to detection using a sample applied to a single PCR analysis cartridge resulting in less sample volume. 
         FIG. 22  illustrates the results of bisulfate conversion using multiple heating steps (bottom panel) compared to a single heating step (top panel). 
         FIG. 23A  illustrates the steps and labor time for a methylation analysis using a standard Qiagen DNA purification kit combined with a Zymo DNA methylation kit (right) compared to a methylation analysis using a Methylation analysis cartridge described herein.  FIG. 23B  shows a comparison of the results obtained using the two different protocols. 
         FIG. 24  shows a comparison of DNA conversion using DABSO as the conversion reagent compared to DNA conversion using the Zymo bisulfite conversion reagent. 
         FIG. 25 , panels A and B, illustrates sensitivity of detection of methylated DNA. Panel A shows a dilution series of methylated DNA (MGMT) (the curves run from the highest concentrations on the left to the lowest concentration on the right. Panel B illustrates the sensitivity of detection of methylated pancreatic cancer markers. 
         FIG. 26  illustrates the results for a reverse complement multiplex assay for both strands (curves from highest to lowest fluorescence: top panel—BNC1_2, BNC1_2, BG, ADAMTS1_1/ADAMTS1_2; bottom panel—BNC1_2, BNC1_2, ADAMTS1_1/ADAMTS1_2, BG, ADAMTS1_1/ADAMTS1_2). 
         FIG. 27A  illustrates the detection of both methylated DNA and mutations in the same cartridge. Top panel illustrates detection of methylated DNA and a Kras G12D mutation in one cartridge, while bottom panel illustrates detection of methylated DNA and wildtype Kras in one cartridge.  FIG. 27B  illustrates detection of methylated DNA and mutations I the same cartridge in two pancreatic cancer cell lines: PANC-1 cells (top panel) and MIA-PaCa cells (bottom panel). 
         FIG. 28  illustrates temperature optimization for multiplex methylation analysis of ADAMTS1, and BNC1 of a forward strand (top) and a reverse strand (bottom) of bisulfite-converted DNA. 
         FIG. 29  illustrates the ability to multiplex the MSP primer and probe sets for BNC1, ADAMTS1, and a control gene ACTB. Probes were combined into two sets based on preferred conditions. 
         FIG. 30  illustrates one set of primers and probes used for detection of MGMT methylation. Internal fwd 22150 (SEQ ID NO: 266); External fwd 22422 (SEQ ID NO: 263); Probe 22419 (SEQ ID NO: 268), Internal rev 22151 (SEQ ID NO: 267); external rev 22423 (SEQ ID NO: 265); template (SEQ ID NO: 1). 
         FIG. 31  shows the results of a comparison between bisulfite pyrsequencing and a MGMT methylation cartridge for extracted DNA (top) and for an FFPET sample (bottom). 
         FIG. 32  illustrates BRCA1 primer and probe set optimization of ΔCt between methylated converted and unmethylated converted DNA. 
         FIG. 33  illustrates a one target assay for BRCA1 methylation tested with the ACTB control gene. As shown, eight different cell lines were tested and the effect of adding NH 4  was compared. 
         FIG. 34  illustrates the results of a three target methylation assay for genes whose methylation is associated with lung cancer (SOX17, CD01, TAC1) in a background of normal plasma and in in three different lung cancer cell lines. 
         FIG. 35  shows the results of a two-cartridge methylation analysis of BNC1 and ACTB. 
         FIG. 36  shows the results of bisulfite conversion analysis of normal urine samples. 
         FIG. 37  shows the results of methylation analysis of normal and cancer sputum samples. 
     
    
    
     DEFINITIONS 
     To facilitate an understanding of the present invention, a number of terms and phrases are defined below: 
     As used herein, the terms “detect”, “detecting” or “detection” may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition. 
     As used herein, the term “detectably different” or “spectrally distinguishable” refers to a set of labels (such as dyes/fluorophores) that can be detected and distinguished simultaneously. 
     DNA methylation DNA methylation refers to the addition of a methyl group (CH3) covalently to the base cytosine (C) typically in the dinucleotide 5′-CpG-3′. The term CpG refers to the base cytosine (C) linked by a phosphate bond to the base guanine (G) in the DNA nucleotide sequence. 
     The term “conversion reagent” refers to a reagent that deaminates cytosine to uracil in single stranded DNA, while leaving 5-MeC essentially unaffected. Illustrative conversion reagents include bisulfites (e.g., sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.) and/or compounds that can produce a bisulfite under appropriate reaction conditions (e.g., DABSO). 
     The phrase “detecting methylation of a gene” generally refers to the detection of methylation of cytosine, typically in CPG islands, in the promoter region of the gene. 
     As used herein, the terms “patient” and “subject” are typically used interchangeably to refer to a human. In some embodiments, the methods described herein may be used on samples from non-human animals, e.g., a non-human primate, canine, equine, feline, porcine, bovine, lagomorph, and the like. 
     As used herein, the terms “oligonucleotide,” “polynucleotide,” “nucleic acid molecule,” and the like, refer to nucleic acid-containing molecules, including but not limited to, DNA. The terms encompass sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. 
     As used herein, the term “oligonucleotide,” refers to a single-stranded polynucleotide typically having fewer than 500 nucleotides. In some embodiments, an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides long. Oligonucleotides may be referred to by their length, for example, a 24 residue oligonucleotide may be referred to as a “24-mer.” 
     As used herein, the term “complementary” to a target gene (or target region thereof), and the percentage of “complementarity” of the probe sequence to the target gene sequence is the percentage “identity” to the sequence of target gene or to the complement of the sequence of the target gene. In determining the degree of “complementarity” between probes used in the compositions described herein (or regions thereof) and a target gene, such as those disclosed herein, the degree of “complementarity” is expressed as the percentage identity between the sequence of the probe (or region thereof) and sequence of the target gene or the complement of the sequence of the target gene that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical as between the 2 sequences, dividing by the total number of contiguous nucleotides in the probe, and multiplying by 100. When the term “complementary” is used, the subject oligonucleotide is at least 90% complementary to the target molecule, unless indicated otherwise. In some embodiments, the subject oligonucleotide is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule. 
     A “primer” or “probe” as used herein, refers to an oligonucleotide that comprises a region that is complementary to a sequence of at least 8 contiguous nucleotides of a target nucleic acid molecule, such as a target gene. In some embodiments, a primer or probe comprises a region that is complementary to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 29, at least 30, at least 319, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 contiguous nucleotides of a target molecule. When a primer or probe comprises a region that is “complementary to at least x contiguous nucleotides of a target molecule,” the primer or probe is at least 95% complementary to at least x contiguous nucleotides of the target molecule. In some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule. 
     The term “nucleic acid amplification,” encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for performing an amplifying step include polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), digital amplification, and the like. Descriptions of such techniques can be found in, among other sources, Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1. 
     In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or, in certain embodiments, can be performed isothermally. 
     The term “hybridize” is typically used herein refer to “specific hybridization” which is the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence, in some embodiments, under stringent conditions. The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target sequence, and to a lesser extent to, or not at all to, other sequences. A “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization are sequence-dependent and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, N.Y. (“Tijssen”). Generally, highly stringent hybridization and wash conditions for filter hybridizations are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m  is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. In certain embodiments very stringent conditions are selected to be equal to the T m  for a particular probe. Dependency of hybridization stringency on buffer composition, temperature, and probe length are well known to those of skill in the art (see, e.g., Sambrook and Russell (2001)  Molecular Cloning: A Laboratory Manual  (3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.). 
     A “sample,” as used herein, generally refers to a biological sample including biological fluids (e.g., blood or blood fractions, serum, plasma, pancreatic juice, cerebrospinal fluid, oral fluid, lymph, intraocular fluid, and the like) and/or tissue samples including, but not limited to biopsy samples, frozen tissue samples, formalin fixed paraffin embedded (FFPE) samples from various tissues including but not limited to breast tissue, endocervical tissue, vaginal tissue, colon/rectal tissue, throat tissue, and other types of human samples, such as blood, stool, and biopsy samples. The term sample also includes diluted and/or buffered forms of the above samples, for example, a buffer into which a swab sample has been placed, a urine sample to which a buffer has been added, and the like. 
     As used herein, the phrase “is indicative of the presence of a cancer or a predisposition to a cancer” means that a particular result tends to indicate that a cancer is present, and/or a precancerous condition is present or likely present. This phrase does not imply a definitive determination that the condition is present. A definitive determination can be made based on further examination or testing that a medical practitioner deems appropriate. Furthermore, this phrase does not require that a determination be made as to which condition may be present based only on the particular result. Rather, it is contemplated that a positive result will be considered in light of other examination or text results to arrive at a differential diagnosis. 
     The term “tubefill procedure” refers to a procedure that is run using standard laboratory instrumentation rather than on a cassette (e.g., rather than with a GENEXPERT®, or modified GENEXPERT® cartridge described herein). 
     DETAILED DESCRIPTION 
     In various embodiments devices and methods are provided that facilitate the rapid detection and/or characterization of methylation in DNA samples. In certain embodiments automated reaction cartridges are provided as are methods that that utilize the automated reaction cartridge(s) to facilitate analysis of the methylation of a DNA sample and, optionally, to measure mRNA levels along with the determination of DNA methylation. In various embodiments the DNA methylation is determined by bisulfite conversion and analysis of the bisulfite converted DNA (e.g., via methylation specific PCR, nucleic acid sequencing, melting point analysis, and the like). In certain embodiments the cartridge performs all or a part of the bisulfite conversion of DNA and all or a part of the analysis of the bisulfite converted DNA. In certain embodiments the cartridge performs all of the steps involved in bisulfite conversion and all or a part of the analysis of the bisulfite-converted DNA. In certain embodiments the cartridge performs all of the steps involved in bisulfite conversion and all of the analysis of the bisulfite-converted DNA. In certain embodiments the cartridge additionally performs an isolation and purification of the DNA to be analyzed. 
     There are several advantages to automating the methylation analysis including for example, reduction in overall processing time, improvements in efficiency, decreased user error and variability, minimization of loss between steps, and an improved ability to use smaller amounts of sample. Use of a cartridge-based process, as described herein, allows for rapid and easy testing of not only multiple sample types but also for evaluating methylation changes observed in several different types of cancers including, but not limited to breast cancer, colorectal cancer, prostate cancer, and lung cancer. 
     The cartridge-based methods described herein additionally permit measurement of mRNA derived from the same sample. Measurement of corresponding upstream and/or downstream mRNA involved in DNA methylation can be important to understand the mechanism and activity of the epigenetic modification. For example, the measurement of DNA methyltransferases (DNMT) mRNA has been studied along with DNA methylation for several cancers (see Table 1). 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Illustrative DNA methyltransferases 
               
               
                 and their importance in particular cancers 
               
               
                 (from Subramaniam et al. (2014) Front Oncol., 4: Article 80, doi: 
               
               
                 10.3389/fonc.2014.00080). 
               
            
           
           
               
               
            
               
                 Methyl- 
                   
               
               
                 transferase 
                 Cancers 
               
               
                   
               
               
                 DNMT1 
                 Leukemia: upregulated-5.3-fold expression 
               
               
                   
                 Gastric cancer-64.8% localized in the cytoplasm and nuclei 
               
               
                   
                 Breast cancer-16.6% 
               
               
                   
                 Hepatocellular carcinoma-100% 
               
               
                   
                 Pancreatic cancer - highly expressed-Gli target gene 
               
               
                   
                 Colon cancer-highly expressed 
               
               
                   
                 Glioblastoma-overexpressed 
               
               
                 DNMT2 or 
                 Hepatocellular carcinoma-reduced expression 
               
               
                 TRDMT1 
                 Colorectal and stomach cancers-lower mRNA expression 
               
               
                 DNMT3A 
                 Acute myeloid leukemia-22.1% mutations 
               
               
                   
                 and affect translation 
               
               
                   
                 Gastric cancer-70.4% localized in the cytoplasm 
               
               
                   
                 Breast cancer-14% 
               
               
                   
                 Hepatocellular carcinoma-60% 
               
               
                   
                 Pancreatic cancer-highly expressed-regulated by Gli 1 
               
               
                   
                 Colon cancer-highly expressed 
               
               
                 DNMT3B 
                 Leukemia: upregulated-11.7-fold expression 
               
               
                   
                 Gastric cancer-51.9% localized in the cytoplasm 
               
               
                   
                 Breast cancer-81.8% poor prognosis 
               
               
                   
                 Breast cancer cell lines-hypermethylation defect resulted in 
               
               
                   
                 aberrant-overexpression DNMT activity 
               
               
                   
                 Hepatocellular carcinoma (60%) and mRNA levels high 
               
               
                   
                 Colon cancer-highly expressed 
               
               
                   
                 Prostate cancer-overexpressed 
               
               
                   
                 Glioblastoma-overexpressed 
               
               
                 DNMT3L 
                 Cervical cancer-promising biomarker 
               
               
                   
                 Embryonal carcinoma-novel biomarker 
               
               
                   
               
            
           
         
       
     
     Often separate independent extractions for DNA or RNA are used for studying and measuring genes and transcripts. Co-detection from the same sample preparation would be ideal to minimize sample preparation, assay to assay, sample-to-sample and cell-to-cell variability. 
     Cartridge-Based Bisulfite Conversion of DNA 
     In certain embodiments the extraction of DNA, bisulfite conversion, and methylation specific PCR are all performed in the cartridge. In one illustrative embodiment, the user will add the sample to a lysis/binding reagent, then mix/vortex the reagent briefly, and then add the sample to a sample port or chamber in the cartridge. Illustrative, but non-limiting lysis reagents (including reagents particularly well suited for FFPE sections) are described in PCT Patent Publication No: WO/2014/052551 (PCT/US2013/061863), which is incorporated herein by reference for the reagents described therein. 
     Additional illustrative lysis reagents for serum or plasma and for formalin-fixed paraffin embedded (FFPE) samples are shown in Example (Tables 13, and 14, respectively). 
     In certain embodiments the cartridge is placed into a processing module and the assay is initiated by clicking through a set of selections within software controlling the processing module (see, e.g.,  FIGS. 11A and 11B ). The cartridge then performs the bisulfate conversion process and analysis of the bisulfate-converted DNA. In certain embodiments mRNA is also determined. While in certain embodiments, all of the operations are performed in the cartridge, in other embodiments, subsets of the various operations are performed in the cartridge as described below. 
     The sample can comprise any biological sample that contains DNA whose methylation state is to be evaluated. Illustrative samples include, but are not limited to isolated DNA and/or isolated total nucleic acids, a cell, a tissue, a biological fluid containing a nucleic acid, and the like. In certain embodiments the biological sample comprises a biological fluid selected from the group consisting of plasma, serum, amniotic fluid saliva, mucus, urine, pancreatic juice, and cerebrospinal fluid. In certain embodiments the sample comprises a tissue sample from a healthy tissue, or a tissue sample from a diseased sample. In certain embodiments the tissue sample is from a fetus, a neonate, a child, an adolescent, or an adult. In certain embodiments the tissue sample comprises tumor cell and/or is derived from a biopsy of a tumor (e.g., a breast cancer, a prostate cancer, a brain cancer, a cervical cancer, an ovarian cancer, a pancreatic cancer, a colon cancer, a gastric cancer, a hepatocellular cancer and the like. In certain embodiments the sample comprises a fixed tissue, e.g., a formalin fixed tissue sample. In certain embodiments the sample comprises an embedded tissue sample (e.g., a formalin-fixed paraffin embedded (FFPE) tissue sample). 
     Bisulfite conversion of DNA typically involves four steps: 
     1) DNA purification; 
     2) DNA denaturation; 
     3) DNA conversion (e.g., bisulfite deamination); and 
     4) Alkali desulphonation. 
     Typically DNA conversion (e.g., using a conversion reagent such as a bisulfite) involves: 1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine; and 2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative. This is followed by Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil as indicated above. 
     As noted above, in certain embodiments, the DNA purification can be performed prior to placing a sample in the cartridge, or alternatively, can be performed by the cartridge itself. Accordingly, in certain embodiments the sample is added directly to the reaction cartridge, while in other embodiments, the sample is mixed with one or more reagents. In certain embodiments DNA preparation typically involves preparing substantially isolated DNA. This may involve lysing cells to release DNA, removing particulates and cellular debris, and/or removing protein components to provide a sample comprising substantially pure nucleic acids (e.g., substantially pure DNA and/or a substantially pure combination of DNA and RNA). In one illustrative, but non-limiting, embodiment, the sample (e.g., a tissue sample) is added to a lysis reagent, agitated and then inserted into the cartridge for further processing. 
     In certain embodiments, all of the reagents necessary to perform bisulfite conversion of the DNA are provided in the cartridge. In certain embodiments, to avoid degradation of reagents over time in the cartridge, certain reagents may be added to the cartridge immediately before use. Thus, for example in certain embodiments, it is contemplated that the cartridge may be loaded with a conversion reagent (e.g., a bisulfite reagent) and/or a guanidium thiocynanate reagent (e.g., GTC-EtOH-Tween) at or about the time the sample is loaded into the cartridge. In certain embodiments, the guanidinium thiocyanate reagent (e.g., GTC-EtOH-Tween) is combined with the sample and added to the cartridge in the sample receiving chamber (e.g., chamber 2 in the GENEXPERT® cartridge). 
     In certain embodiments when performing the bisulfite conversion of DNA using a reaction cartridge (e.g., GENEXPERT® cartridge), the method comprises 
     i) contacting a biological sample comprising a nucleic acid to a first matrix material comprising a first column or filter where said matrix material binds and/or filters nucleic acids in said sample and thereby purifies the DNA; 
     ii) eluting the bound DNA from the first matrix material (e.g., using an alkaline solution) and denaturing the DNA to produce eluted denatured DNA; 
     iii) heating the eluted DNA in the presence of a conversion reagent (e.g., a reagent that provides bisulfite ions) to produce a converted (e.g., a deaminated) nucleic acid; 
     iv) contacting the converted nucleic acid to a second matrix material comprising a second column to bind said deaminated nucleic acid to said second matrix material (note in certain embodiments the second column can be a column different than the first column, or in other embodiments, the same column used a second time); 
     v) desulphonating the bound deaminated nucleic acid and/or simultaneously eluting and desulphonating the nucleic acid by contacting the deaminated nucleic acid with an alkaline solution to produce a converted (e.g., bisulfite converted) nucleic acid; and 
     vi) eluting the converted nucleic acid from said second matrix material, wherein at least steps iv) through vi) are performed in a one reaction cartridge. 
     In certain embodiments the method further includes the analysis of the converted DNA. Accordingly, in certain embodiments, the method further comprises: 
     vii) performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on the converted nucleic acid to determine the methylation of the nucleic acid, wherein at least steps iv) through vi) are performed in a single reaction cartridge. 
     In certain embodiments at least steps iii) through vi) are performed in one reaction cartridge. 
     In certain embodiments at least steps ii) through vi) are performed in one reaction cartridge. 
     In certain embodiments at least steps i) through vi) are performed in one reaction cartridge. 
     In certain embodiments at least steps i) through vii) are performed in one reaction cartridge. 
     It is noted that the first column and, where present, the second column can refer to discrete columns. However, particularly when integrated into a reaction cartridge, the “column” can simply be a matrix material disposed in a chamber or channel in the cartridge. In various embodiments the “columns” act as filters and/or as affinity columns that bind nucleic acids. Accordingly, in certain embodiments the column contains a matrix material that binds a nucleic acid (e.g., DNA and/or RNA). Illustrative matrix materials include, but are not limited to, glass (silica), an ion exchange resin, hydroxyapatite, and the like. It will be recognized that the matrix materials can take a number of forms. Thus, in certain embodiments, the matrix material comprises a fibrous material a particulate material (e.g., microbeads, nanobeads, etc.), a structured material (e.g., porous “baffle” system”, a serpentine channel, and the like). In certain embodiments the first column and second column are different columns (chambers or channels). In other embodiments the first column and the second column are the same column (chamber or channel) that is used twice (e.g., a first time and a second time). 
     In certain embodiments, the use of one or more additional filters, e.g., to clean up the initial sample prior to contacting with the first matrix material, is contemplated. Thus, for example, in certain embodiments, a filter matrix (e.g., polycarbonate filter, nylon filter, polypropylene filter, polyester filter, nylon filter, ceramic filter, polytetrafluoroethylene filter, and the like) is disposed in the sample receiving chamber or “downstream” from the sample receiving chamber and before the first “column”. It is also recognized, that in certain embodiments, the sample, can be lysed and/or filtered prior to deposition into a sample receiving chamber. 
     In certain illustrative ,but non-limiting embodiments, the methods described herein can be performed using a GENEXPERT® cartridge (Cepheid, Inc., Sunnyvale, Calif.) or a variant thereof. In various embodiments sample extraction, and/or amplification, and/or DNA conversion, and/or detection can all be carried out within this self-contained “laboratory in a cartridge” (see, e.g.,., U.S. Pat. Nos. 5,958,349, 6,403,037, 6,440,725, 6,783,736, and 6,818,185, each of which is herein incorporated by reference in its entirety). In various embodiments components of the cartridge can include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection (e.g., of PCR amplification products). A reaction tube can be provided that permits very rapid heating and/or thermal cycling. 
     In certain embodiments an illustrative GENEXPERT® cartridge comprises a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with the central valve assembly where the central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with the central valve. Rotation of the valve assembly determines which chamber are in fluid communication with the central valve. One illustrative GENEXPERT® cartridge is illustrated in  FIG. 1A  which show the cartridge, processing/reagent chambers, a reaction tube (e.g., heating and/or thermocycling tube), optional optical windows, and a valve that facilitates fluid transfer from chamber to chamber. 
     An illustrative layout of the cartridge is shown in  FIG. 1B  which provides a top view of the cartridge identifying various chambers by number. In one illustrative, but non-limiting embodiment, the components of the chambers comprising the cartridge are as listed in Table 2. It will be recognized that this disposition of reagents and chamber is illustrative and non-limiting. Using the teachings provided herein other reagent dispositions and/or other chamber configurations will be available to one of skill in the art. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 One illustrative embodiment showing chamber contents for use of a 
               
               
                 GENEXPERT ® cartridge for measurement of DNA methylation. 
               
            
           
           
               
               
               
            
               
                 Chamber 
                 Chamber 
                 Initial Volume 
               
               
                 # 
                 Contents 
                 (μL) 
               
               
                   
               
               
                  1 
                   
                 — 
               
               
                   2* 
                 Sample chamber (sample mixed with 
                   
               
               
                   
                 e.g., GTC-EtOH-Tween precipitation 
                   
               
               
                   
                 reagent) 
                   
               
               
                    3** 
                 GTC-EtOH 
                   
               
               
                     4*** 
                 Bisulfite reagent 
                   
               
               
                   
                 (e.g., 8M ammonium bisulfite) 
                   
               
               
                  5 
                 Buffer 
                   
               
               
                   
                 e.g., 50 mM Tris pH 8.5 
                   
               
               
                  6 
                   
                 — 
               
               
                  7 
                   
                 — 
               
               
                  8 
                 Rinse (e.g., PEG 200) 
                   
               
               
                  9 
                 Beads (e.g., polymerase, primer, probe) 
                   
               
               
                 10 
                 Elution/Desulphonation reagent 
                   
               
               
                   
                 (e.g., 15 mM KOH) 
                   
               
               
                 11 
                 Beads (e.g., polymerase, primer, probe) 
               
               
                   
               
               
                 *Sample is added to chamber 2 by user 
               
               
                 **In certain embodiments, GTC-EtOH is added at time of use (e.g., when sample is added). In certain embodiments GTC-EtOH is provided as reagent already disposed in cartridge. 
               
               
                 ***In certain embodiments, bisulfite reagent is added at time of use (e.g., when sample is added). In certain embodiments bisulfite reagent is already disposed in cartridge. 
               
            
           
         
       
     
     One embodiment of a step-by-step workflow for the determination of DNA methylation utilizing such a cartridge is shown in  FIG. 1C . In this cartridge configuration, there are five chambers that, in use (e.g., when the cartridge is operating to determine DNA methylation), will hold reagents and buffers (e.g., chambers 3, 4, 5, 8, and 10), one chamber that will hold the sample added by the user (e.g., chamber 2), and one or two (or more) chambers holding analysis reagents (e.g., MSP reagents such as enzyme reaction, template specific reaction, and/or or 200 mM Tris pH 7.0, e.g., as beads) (e.g., chambers 9, and 11). In certain embodiments, the reagents (e.g., polymerase, reverse transcriptase, primer, probe) are provided in solution. In certain embodiments the reagents are provided as lyophilized powders. In certain embodiments the reagents are provided as lyophilized beads. The beads can further comprise agents to that improve reagent stability and/or activity (see, e.g., U.S. Patent Publication No: 2006/0068399 which is incorporated herein by reference for the beads, bead fabrication, and bead formulations described therein. 
     In certain embodiments the cartridge, as provided contains all of the reagents necessary to run the cartridge and only the sample (e.g., sample in buffer/lysis/precipitation solution) is added to the cartridge. In certain embodiments the cartridge is provided without the GTC-EtOH and/or the bisulfite reagents and one or both are added at the time of use. Thus, in certain embodiments, the GTC-EtOH reagent is added to the cartridge at the time of use, in certain embodiments the bisulfite reagent (in addition to the sample) is added to the chamber at the time of use, and in certain embodiments, both the GTC-EtOH and the bisulfite reagent (in addition to the sample) are added to the cartridge at the time of use. In certain embodiments these reagents are added directly to the desired chambers (see, e.g., Table 2). In certain embodiments ports are provided for loading the reagents and the ports are configured to deliver the reagent(s) to the desired chambers. 
     At the start of the assay, the cartridge dispenses the sample, e.g. from chamber 2 over a glass fiber column (e.g. the first column) in the cartridge. DNA is eluted off the column and simultaneously denatured by an alkali solution, e.g., a low concentration of potassium hydroxide from chamber 10 into a concentrated bisulfite reagent (e.g., concentrated ammonium bisulfite) in Chamber 4. In certain embodiments the DNA is eluted with an alkaline solution of KOH with a pH greater than about 10.5, or a pH greater than about pH 12. In certain embodiments the DNA is eluted with 10-15 mM KOH. 
     As indicated above, the DNA is eluted (optionally with a burst of sonication) into the bisulfite reagent. In various embodiments the conversion reagent (e.g., bisulfite reagent) is present at a concentration ranging from about 4 M to about 10 M, or from about 5 M to about 8 M, or from about 6 M or about 7 M. In certain embodiments the bisulfite solution comprises sodium metabisulfite, or potassium bisulfite, or ammonium bisulfite, or cesium bisulfite, or DABSO (1,4-diazoniabicyclo[2.2.2]octane-1,4-disulphinate, see, e.g.,  FIG. 16 ). In certain embodiments the conversion reagent (e.g., bisulfite reagent) contains radical scavengers, including, but not limited to one or more chemicals to prevent sulfite oxidation to sulfate (TROLOX and hydroquinone), and/or catalysts (polyamines). 
     The DNA-bisulfite (DNA/conversion reagent) mix is then introduced into a temperature controlled chamber or channel and incubated at a temperature ranging from about 40° C. to about 95° C. In certain embodiments the mix is incubated at a constant temperature, while in other embodiments, e.g., where the temperature controlled chamber or channel is a thermocycling chamber or channel (e.g., a smartcycler tube in the back of the cartridge), the mix is thermally cycled (e.g., between 60° C. and 95° C.). The mix is incubated until the DNA is converted (e.g., deaminated). In certain embodiments the incubation is for a period of time that ranges from about 5 minutes up to about 4 hours, or preferably from about 15 minutes up to about 45 minutes. 
     Following incubation the DNA/conversion reagent) (e.g., DNA-bisulfite) solution is mixed with fresh guanidinium thiocyanate-EtOH, e.g., from chamber 3 and dispensed over a matrix material. In certain embodiments the first column is reused, hence there is only one column and the second column and the first column are the same. In certain embodiments the second column is a separate column different than the first column. 
     The DNA bound to the second column matrix material is washed with fresh GTC-EtOH (e.g., from chamber 3) and rinsed (e.g., with a PEG 200 rinse, e.g., from chamber 8). The DNA is then desulphonated on the column, or is simultaneously eluted and desulphonated by contacting the deaminated nucleic acid with an alkaline solution (e.g., KOH from chamber 10 to produce a bisulfite converted nucleic acid. In certain embodiments the incubation is for a period of time ranging from about 1 minute to about 1 hour, or from about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes, or for about 15 minutes. 
     Where the initial incubation was in a thermocycling chamber that is to be further used, the thermocycling chamber or channel is washed with a buffer to remove residual bisulfite and neutralize pH. It was a surprising discovery that incubation with a conversion reagent (e.g., a bisulfite reagent), and/or desulphonation can be performed in a channel or chamber that is later used for PCR without bisulfite contamination substantially interfering with the later PCR reaction(s). 
     The eluted desulphonated bisulfite-converted DNA can be mixed with an appropriate buffer and analyzed for methylation. In certain embodiments the converted DNA is mixed with concentrated Tris, enzyme reaction, and template specific beads (e.g., beads comprising primers and/or probes for the PCR or nested PCR reaction(s)) in chambers 9 and 11, and the final mixture is aspirated into the thermocycling tube or chamber for the methylation specific quantitative PCR reaction. 
     Bisulfite contamination during the qPCR step can be the primary failure mode of the methylation cartridge. Residual bisulfite can result from either direct contamination of the PCR reaction tube (e.g., during the bisulfite conversion step) or from indirect contamination (e.g. cross contamination during bisulfite fluidic movements between chambers). Residual bisulfite contamination, if present, can be measured by bisulfite-mediated probe cleavage during the qPCR step, which results in an increase in fluorescence during the earlier qPCR cycles (cycles 1-10) typically used for background subtraction. Accordingly, in certain embodiments, the cartridge comprises beads that provide one or more probes that are cleavable during PCR if bisulfite is present. Results of a run containing bisulfite contamination are shown in  FIG. 14 . 
     While the methods above (and in Example 4, see, e.g.,  FIG. 13A ) are described with respect to specific chambers in the GENEXPERT® cartridge, it will be recognized that the particular reagent/chamber assignments can be varied depending on the particularities of the methylation analysis protocol applied. 
     Thus, for example, operation of a methylation analysis cartridge (e.g., a GENEXPERT® cartridge can be generally described by a flow chart (see, e.g.,  FIGS. 1C and 13B ). In the illustrative, but non-limiting embodiment shown in  FIG. 13B , the DNA sample is provided in a binding buffer (e.g., a buffer comprising GTC-EtoH, in certain embodiments after the sample is processed with proteinase K and/or a lysis solution). In certain embodiments the sample is obtained from a sample preparation cartridge as described herein (see, e.g.,  FIG. 20 ). 
     The sample in binding buffer is introduced into a sample receiving chamber of the cartridge. In operation the cartridge is operated to deliver the sample solution to a matrix (“column”) that binds the DNA. The bound DNA is then eluted from the column using an alkaline reagent (e.g., KOH solution) combined with a bisulfite reagent and moved to a heating tube (typically the PCR reaction tube) in the cartridge where the bisulfite reaction proceeds (e.g., at about 50° C. or about 60° C. to about 90° C. for about 45 minutes (or up to about 90 minutes), in this illustrative protocol). The reacted DNA is combined with a binding buffer (e.g., 2.25 M Guanidinium thiocyanate, 22.5 mM Tris pH 7.0, 0.5% Tween20, 50% Ethanol, and 0.005% SE-15 antifoam (a 10% emulsion of an active silicon antifoam and non-ionic emulsifiers)) and moved back to the same column, or to a different column, where it again binds to the column matrix. The reacted DNA is washed with GTC-EtOH, rinsed with PEG (e.g., PEG200) and eluted again from the column using an alkaline reagent (e.g., KOH) which also desulphonates the DNA. While the DNA is desulphonating the reaction tube (e.g., PCR reaction tube) can be heated and rinsed (e.g., 10× rinse) to remove any bisulfite reagent. The eluted DNA (or a portion thereof) can be moved to a reaction tube for PCR and/or nested PCR. 
     It will be appreciated that these operations can be performed on the entire sample or on a portion of the DNA sample. In the latter case a portion of the sample can be stored in one or more chambers and used as a control, or subjected to a different analysis/protocol. 
     Co-Purification and Detection of both RNA Expression and DNA Methylation. 
     In certain embodiments methods for co-purification and detection of both altered RNA expression of genes along with DNA methylation (MSP) in a cartridge-based assay (e.g., utilizing a GENEXPERT® cartridge) are provided. In certain embodiments these assays would identify altered expression of e.g. DNMT correlated with tumor-specific methylation from the same sample preparation. In certain embodiments these assays can be used to verify expression and methylation status. 
     We have shown that we can elute nucleic acids off the column using a Tris buffered elution that retains a portion of nucleic acids on the column. In one illustrative embodiment, an RNA fraction is eluted and retained, e.g., in a chamber in the cartridge using a Tris solution. 
     After saving the RNA fraction, NaOH or KOH elution which will strip the column and elute and denature the DNA which would go into bisulfite for conversion as described above. Then, ether using the RNA elution fraction to elute the bisulfite converted DNA from the column or using the KOH elution mix the two fractions (RNA and converted DNA products) are mixed for RNA plus bisulfite converted qRT-PCR. This involves incorporating a reverse transcriptase (RT) step for the RNA plus MSP (or other analytic method) in the same tube from the same sample. Alternative methods include, but are not limited to performing the RT step independently prior to mixing with DNA (combine cDNA and DNA) for qPCR, or PCR for DNA or RT RNA could be done independently/serially using one thermocycling tube/chamber or simultaneously using multiple thermocycling tubes/chambers in the cartridge. 
     Analysis of Converted DNA 
     Numerous analytic methods can be performed in the cartridge to evaluate DNA methylation. Alternatively, in certain embodiments, the cartridge can be coupled to another device and/or system for further analysis of the converted (e.g., bisulfite or DABSO converted) DNA. Illustrative methods include, but are not limited to methylation specific PCR (MSP), direct sequencing, high resolution melting analysis (HRM), pyrosequencing (sequencing by addition), base-specific cleavage analysis (e.g. base-specific MALDI-TOF), and the like. 
     Methylation-Specific PCR (MSP). 
     In various embodiments methylation-specific PCR can be used to evaluate methylation status of the target DNA. MSP utilized primer and/or probe sets designed to be “methylated-specific” by including sequences complementing only unconverted 5-methylcytosines, or, on the converse, “unmethylated-specific”, complementing thymines converted from unmethylated cytosines. Methylation is then determined by the ability of the specific primer to achieve amplification. This method is particularly effective for interrogating CpG islands in regions of high methylation density, because increased numbers of unconverted methylcytosines within the target to be amplified increase the specificity of the PCR. In certain embodiments placing the CpG pair at the 3′-end of the primer also improves the specificity. 
     In certain embodiments methylation is evaluated using a MethyLight method. The MethyLight method is based on MSP, but provides a quantitative analysis using quantitative PCR (see, e.g., Eades et al. (2000)  Nucleic Acids Res.,  28(8): E32. doi:10.1093/nar/28.8.e32). Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region. In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. Quantitation can be made in reference to a methylated reference DNA. One modification to this protocol to increase the specificity of the PCR for successfully bisulphite-converted DNA (ConLight-MSP) uses an additional probe to bisulphite-unconverted DNA to quantify this non-specific amplification (see, e.g., Rand et al. (2002)  Methods  27(2): 114-120). 
     In various embodiments the MethyLight methods utilize TAQMAN® technology, which is based on the cleavage of a dual-labeled fluorogenic hybridization probe by the 5′ nuclease activity of Taq-polymerase during PCR amplification (Eads et al. (1999)  Cancer Res.,  59: 2302-2306; Livak et al. (1995)  PCR Meth. Appl.,  4: 357-362; Lee et al. (1993)  Nucleic Acids Res.,  21: 3761-3766; Fink et al. (1998)  Nat. Med.,  4: 1329-1333). The use of three different oligonucleotides in the TAQMAN® technology (forward and reverse PCR primers and the fluorogenic hybridization probe) offers the opportunity for several sequence detection strategies. 
     For example, the sequence discrimination can occur at the level of the PCR amplification process (see, e.g.,  FIG. 4A , panel C) and/or at the level of the fluorogenic probe hybridization (see, e.g.,  FIG. 4A , panel B). In both steps, the discrimination is based on the differential annealing of the perfectly matched, versus mismatched oligonucleotides. In the MethyLight technology, sequence discrimination at the PCR amplification level occurs by designing the primers and probe, or just primers, or just probes, to overlap potential sites of DNA methylation (e.g., CpG dinucleotides). One approach is simply a fluorescence-based version of the MSP technique (Herman et al. (1996)  Proc. Natl. Acad. Sci. USA,  93: 9821-9826). Each oligonucleotide (primers and probe) can cover anywhere from zero to multiple CpG dinucleotides. Each CpG dinucleotide can result in two different sequence variations following bisulfate conversion, depending on whether that particular site was methylated (mCpG) or unmethylated (UpG). For example, if an oligonucleotide overlaps two CpG dinucleotides, then the number of possible sequence variants in the genomic DNA within the region covered by that oligonucleotide is 2×2=4. If both of the primers and the probe each overlap two CpGs, then the total number of variants contained within the sequence covered by the oligonucleotides is 4×4×4=64. In theory, one could design separate PCR reactions to analyze the relative amounts of each of these potential 64 sequence variants. However, significant methylation information can be derived from the analysis of a much smaller number of variants by designing reactions for the fully methylated and fully unmethylated molecules, which represent the two most extreme sequence variants this hypothetical example. The ratio between these two reactions or the ratio between the methylated reaction and a control reaction provides a measure of the prevalence of methylated molecules at this locus. 
     The MethyLight technology can also be modified to avoid sequence discrimination at the PCR amplification level. If the neither the primers nor the probe overlie any CpG dinucleotides, then the reaction represents unbiased amplification and can serve as a control for the amount of input DNA. One illustrative useful control reaction is one in which the entire amplicon is devoid of any CpG dinucleotides in the unconverted genomic sequence. When just the probe is designed to cover CpG dinucleotides, then sequence discrimination occurs solely at the level of probe hybridization. In this version, all sequence variants resulting from the sodium bisulfate conversion step are amplified with equal efficiency, as long as there is no amplification bias (see, e.g., Wamecke et al. (1997)  Nucleic Acids Res.,  25: 4422-1426). In this case, the design of separate probes for each of the different sequence variants associated with a particular methylation pattern (2×2=4 probes in the case of two CpGs) allows a quantitative determination of the relative prevalence of each sequence permutation in the mixed pool of PCR products. 
     In certain embodiments the analysis methods also provide PCR specific for unconverted DNA. This PCR may interrogate SNPs, mutations, and/or translocations, etc. In this regard, it is noted that the detection of mutations and methylation in a single cartridge is illustrated in Example 12 (see, e.g.,  FIGS. 27A and 27B ). Detection of SNPs, mutations, translocations and the like can readily be accomplished by the inclusion of primers and probe sets specific for the detection of these targets. 
     Nested PCR and Multiplex PCR Assays. 
     In certain embodiments methylated DNA can be detected using an PCR methods well known to those of skill in the art. In certain embodiments a nested PCR reaction is used to detect methylation targets. In one illustrative, but non-limiting, embodiment (see, e.g., Example 4), a nested PCR protocol can be used where the first 15-20 cycle PCR reaction is not specific for methylation but only the converted DNA sequences (i.e., they do not cross CpGs or in instances when they do a R=purine or Y=pyrimidine is used to catch both methylated and unmethylated template sequences). The second qPCR reaction (e.g., a 45 cycle qPCR reaction) can contain both primers and probes that are specific for typically 2-3 methylated CpGs. 
     It will be noted that in certain embodiments, a MethyLight analysis is performed using a single probe (see, e.g.,  FIG. 4B ). In this approach, using a single, e.g., methylation-specific, probe (PR3) along with methylation specific forward (FW) and reverse (RV) primers, methylation specific PCR for the probe (PR3) provides a signal that is dependent on methylation and bisulfite conversion for the FW, RV and PR3 sequences. 
     In various embodiments, multiplexed PCR assays are contemplated. By way of illustration,  FIG. 4C  illustrates a MethyLight approach using multiple probes (PR1, PR2, . . . PR5) that each target different regions. The combined signal from all the probes (PR1, PR2, PR3, PR4, and PR5) yields a measure of the amount/degree of methylation. In certain embodiments each probe has its own specific dye/fluor so that it is detectable independently of the other probes. Thus, even where one target is not methylated, a signal may still be detected, e.g., if PR3 is not methylated there will be no/less signal from the remaining probes.  FIG. 4D  illustrates a MethyLight approach using multiple probes (PR1 . . . PR5) that each target the same region, but provide signals for different methylation patterns. While the approach illustrated in  FIG. 4C  can provide detection from a larger region, this multi-probe approach on a single smaller region could be accomplished with sequence specific primers or probes interrogating the extent of methylation across a specific sequence after bisulfite conversion. 
     In certain embodiments a reverse complement multiplex assay for both strands can be used (see, e.g.,  FIG. 26 ). Following bisulfite conversion, both strands lose their complementarity. Thus, primer and probe sets can be designed for one strand or the other, and result in unique amplicons. In addition to providing “more opportunities” for detection, this approach can potentially help with sensitivity (at LOD, if only one strand or the other ends up in the tube, this approach would ensure the signal gets picked up). This approach permits the multiplex assay to be expanded to detect different CpGs at the same promoter site. The reverse complement multiplex provides more opportunities to detect target methylation and to pick up heterogenous methylation. 
     The foregoing methods are illustrative and non-limiting. Using the teachings provided herein numerous variations of MSP and/or MethyLight analysis will be available to one of skill in the art and implementable on a reaction cartridge, e.g. as described herein. 
     Direct Sequencing 
     In certain embodiments methylation status of the DNA can be determined using direct sequencing methods. In certain embodiments, the method can utilize PCR and standard dideoxynucleotide DNA sequencing to directly determine the nucleotides resistant to bisulphite conversion (see, e.g., Frommer et al. (1992)  Proc. Natl. Acad. Sci. USA,  89 (5): 1827-1831). In various embodiments primers are designed to be strand-specific as well as bisulphite-specific (e.g., primers containing non-CpG cytosines such that they are not complementary to non-bisulphite-treated DNA), flanking (but not involving) the methylation site of interest. Therefore, it will amplify both methylated and unmethylated sequences, in contrast to methylation-specific PCR. All sites of unmethylated cytosines are displayed as thymines in the resulting amplified sequence of the sense strand, and as adenines in the amplified antisense strand. In certain embodiments nested PCR methods can be used to enhance the product for sequencing. 
     In certain embodiments the sequencing can be performed in the cartridge. In other embodiments, the cartridge can be coupled (e.g., fluidic coupled) to a sequencing machine to provide the sequencing analysis. Alternatively, in certain embodiments, the amplified product can be manually transferred from the cartridge to the sequencing system. 
     High Resolution Melting Analysis (HRM) 
     In certain embodiments high-resolution melting analysis (HRM) can be used to differentiate converted from unconverted bisulphite-treated DNA. HRM is a quantitative PCR technique in which the PCR amplicons are analyzed directly by temperature ramping and resulting liberation of an intercalating fluorescent dye during melting (see, e.g., Wojdacz and Dobrovic (2007)  Nucleic Acids Res.  35(6): e41). The degree of methylation, as represented by the C-to-T content in the amplicon, determines the rapidity of melting and consequent release of the dye. This method allows direct quantitation, but assesses methylation in the amplified region as a whole rather than at specific CpG sites. 
     Pyrosequencing 
     In certain embodiments pyrosequencing (sequencing by synthesis) can be used to analyze bisulphite-treated DNA without using methylation-specific PCR (see, e.g., Colella et al. (2003).  BioTechniques  35(1): 146-150; Tost et al. (2003)  BioTechniques  35(1): 152-156; and the like). Sequencing by synthesis differs from Sanger sequencing in that it utilizes the detection of phosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides. The DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that typically only one out of four of the possible A/T/C/G nucleotides are added and available at a time so that only one letter can be incorporated on the single stranded template (which is the sequence to be determined). 
     Following PCR amplification of the region of interest, pyrosequencing can be used to determine the bisulphite-converted sequence of specific regions (e.g., CpG sites). In certain embodiments the ratio of C-to-T at individual sites can be determined quantitatively based on the amount of C and T incorporation during the sequence extension. 
     A modification of this technique can utilize allele-specific primers that incorporate single-nucleotide polymorphisms (SNPs) into the sequence of the sequencing primer(s) , thus allowing for separate analysis of maternal and paternal alleles (see, e.g., Wong et al. (2006)  BioTechniques  41(6): 734-739). This modification is particularly of use for genomic imprinting analysis. 
     Base-Specific Cleavage Analysis. 
     In certain embodiments, base-specific cleavage/MALDI-TOF takes advantage of bisulphite-conversions by adding a base-specific cleavage step to enhance the information gained from the nucleotide changes (Ehrich et al. (2005)  Proc. Natl. Acad. Sci. USA,  102 (44): 15785-15790). By first using in vitro transcription of the region of interest into RNA (by adding an RNA polymerase promoter site to the PCR primer in the initial amplification), RNase A can be used to cleave the RNA transcript at base-specific sites. RNase A cleaves RNA specifically at cytosine and uracil ribonucleotides and base-specificity is achieved by adding incorporating cleavage-resistant dTTP when cytosine-specific (C-specific) cleavage is desired, and incorporating dCTP when uracil-specific (U-specific) cleavage is desired. The cleaved fragments can then be analyzed by MALDI-TOF or other methods. Bisulphite treatment results in either introduction/removal of cleavage sites by C-to-U conversions or shift in fragment mass by G-to-A conversions in the amplified reverse strand. C-specific cleavage will cut specifically at all methylated CpG sites. By analyzing the sizes of the resulting fragments (e.g., using MALDI-TOF, capillary electrophoresis, microchip electrophoresis, and the like), it is possible to determine the specific pattern of DNA methylation of CpG sites within the region, rather than determining the extent of methylation of the region as a whole. 
     Methylation-Sensitive Single-Strand Conformation Analysis (MS-SSCA). 
     Methylation -sensitive single strand conformation analysis (MS-SSCA) is based on the single-strand conformation polymorphism analysis (SSCA) method developed for single-nucleotide polymorphism (SNP) analysis (Bianco et al. (1999)  Hum. Mutat.  14(4): 289-293). SSCA differentiates between single-stranded DNA fragments of identical size but distinct sequence based on differential migration in non-denaturating electrophoresis. In MS-SSCA, this is used to distinguish between bisulphite-treated, PCR-amplified regions containing the CpG sites of interest. Although SSCA lacks sensitivity when only a single nucleotide difference is present, bisulphite treatment frequently makes a number of C-to-T conversions in most regions of interest, and the resulting sensitivity can be high. In certain embodiments MS-SSCA can also provide semi-quantitative analysis of the degree of DNA methylation based on the ratio of band intensities. Typically, however, MS-SSCA assesses all CpG sites as a whole in the region of interest rather than individual methylation sites. 
     Methylation Targets. 
     As noted above, DNA methylation is of interest in a wide number of contexts. In certain embodiments, the amount of DNA methylation is of clinical interest particularly in oncology. Aberrant DNA methylation patterns (hypermethylation and hypomethylation compared to normal tissue) have been associated with a large number of human malignancies. Hypermethylation typically occurs at CpG islands in the promoter region and is associated with gene inactivation. A lower level of leukocyte DNA methylation is associated with many types of cancer (Zhang et al. (2011) Epigenetics, 6(3): 293-299). Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms. Typically, there is hypermethylation of tumor suppressor genes and hypomethylation of oncogenes (see, e.g., Lund et al. (2004)  J. Biol. Chem.  279(28): 29147-29154). 
     In this regard, it is noted that DNA methylation provides a prognostic indicator for Stage I Non-Small-Cell Lung Cancer (NSCLC). In particular, it was discovered that hypermethylation of five genes was significantly associated with shorter relapse-free survival (RFS) in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (see, e.g., Sandoval et al. (2013)  J. Clin. Oncol.,  4140-4147). 
     Similarly it has been observed that malignant gliomas may have the MGMT gene inactivated due to methylation of its promoter region. The prediction, born out by current research, is that by methylating the MGMT gene, a better response to chemotherapy can occur (as the tumor has no means to repair the DNA damage induced by the alkylating agent). In gliomas, MGMT promoter methylation is a favorable prognostic marker in the setting of either radiation or chemotherapy (see, e.g., //neurosurgery.ucsd.edu/brain-tumor-research-mgmt/). 
     By way of further illustration, Table 3 illustrates various genes that are hypermethylated in certain cancers. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 shows illustrative, but non-limiting examples of genes 
               
               
                 hypermethylated in sporadic cancers 
               
               
                 (see, e.g., Baylin (2005)  Nature Clinical Practice Oncology , 2: S4-S11). 
               
            
           
           
               
               
            
               
                 Gene or gene product 
                 Tumor type 
               
               
                   
               
               
                 Rb 
                 Retinoblastoma 
               
               
                 APC 
                 Colorectal and other cancers 
               
               
                 p14/ARF 
                 Colorectal cancer 
               
               
                 p15/CDKN2B 
                 Leukemias 
               
               
                 p16/CDKN2A 
                 Various cancers 
               
               
                 BRCA1 
                 Breast, ovarian cancer 
               
               
                 VHL 
                 Renal cell cancers 
               
               
                 hMLH1 
                 Colorectal, gastric, endometrial cancers 
               
               
                 ER-α 
                 Breast, colorectal, other cancers 
               
               
                   
               
            
           
         
       
     
     In various illustrative, but non-limiting, embodiments measurement of methylation of any one of more of the promoters of the following genes is contemplated: APC, ARF, CDKN2B, CDKN2A, BRCA1, VLH, hMLH1, MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, AKR1B1, HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. 
     Pancreatic Cancer. 
     In certain embodiments methylation status is determined for one or more promoters where methylation status is a marker for the presence and/or prognosis of pancreatic cancer. It was determined that the frequency of methylation of one or more of ADAMTS1, or BNC1, can be used to detect and/or stage pancreatic cancer. Thus, illustrative, but non-limiting methylation markers for pancreatic cancer include, but are not limited to ADAMTS1 and/or BNC1. Illustrative primers and probes for the detection of methylation at the promoters of these genes are shown in Table 4, below (referencing Table 5 for particular sequences), and in Table 12 in Example 4). In certain embodiments primers and probes are provided for the detection of methylation in the forward strand of the converted DNA and/or for the detection of methylation in the reverse strand of the converted DNA. 
     Breast Cancer. 
     In certain embodiments methylation status is determined for one or more promoters where methylation status is a marker for the presence and/or prognosis of breast cancer. Illustrative methylation markers for breast cancer include, but are not limited to RASSF1A, and/or AKR1B1, and/or HOXB4, and/or HIST1H3C, and/or RASGRF2, and/or TM6SF1. Illustrative primers and probes for the detection of methylation at the promoters of these genes are shown in Table 4, below (referencing Table 5 for particular sequences), and in Table 11 in Example 4. 
     In certain embodiments methylation status is determined for one or more promoters where methylation status is a marker for the presence or likelihood of lung cancer. Illustrative methylation markers for lung cancer include, but are not limited to CDO1, SOX17, TAC1, and/or HOXA7. 
     The methods described herein are not limited to determining methylation of the promoters of these genes. Using the methods described herein methylation of essentially any target of interest is possible. 
     It will be noted, however that measurement of DNA methylation need not be limited to measurement of methylation at CPG islands in promoters. For example, it has been demonstrated that gene body methylation can also alter gene expression and can provide a therapeutic target in cancer (see, e.g., Yang et al. (2014) Cancer Cell, 26(4): 577-590). 
     Additionally, measurement of DNA methylation has prognostic/therapeutic applications for pathologies other than cancer. For example, aberrant methylation on regions on chromosomes 13, 18, 21, X, and Y can be used to diagnose Down syndrome (see, e.g., Patsalis et al. (2012)  Exp. Opin. Biol. Ther.  12(Suppl. 1): S155-S161). Because fetal DNA and maternal DNA are differentially methylated, cell-free DNA in maternal plasma can provide a source of fetal DNA, which can be obtained non-invasively and utilized to assess the methylation state of the aforementioned chromosomes (or other chromosomes or genes). 
     As noted above, in certain embodiments, the cartridges and methods described herein are also used to determine mRNA levels, e.g., to determine expression of various methyltransferases. In certain embodiments, expression level of RNA is determined for a methyltransferase selected from the group consisting of DNMT1, DNMT2, DNMT3A, DNMT3B, and TNMT3L. 
     Primers/Probes and Multiplex Analysis 
     In various embodiments the methods described herein can involve nested PCR reactions and the cartridges described herein can contain reagents (e.g., primers and probes) for such nested PCR reactions. For example, in certain embodiments, methylation is detected for one, two, three, four, five, or six genes (gene promoters). Since bisulfite conversion of a DNA changes cytosine resides to uracil, but leave 5-methyl cytosine residues unaffected, the forward and reverse strands of converted (bisulfite-converted) DNA are no longer complementary. Accordingly, it is possible to interrogate the forward and revers strands independently (e.g., in a multiplex PCR reaction) to provide additional specificity and sensitivity to methylation detection. In such instances, assaying of a single target can involve a two-plex multiplex assay, while assaying of two, three, four, five, or six target genes can involve four-plex, six-plex, 8-plex, 10-plex, or 12-plex multiplex assays. In certain embodiments the assays can be divided into two multiplex reactions, e.g., to independently assay forward and reverse strands. However, it will be recognized that when split into multiple multiplex assays, the grouping of assays need not be by forward or reverse, but can simply include primer/probe sets that are most compatible for particular PCR reaction conditions. 
     As indicated above, numerous cancers can be identified, and/or staged and/or a prognosis therefor determined by the detection/characterization of the methylation state on the forward and/or reverse strand of gene promoters whose methylation (or lack thereof) is associated with a cancer. Illustrative gene (promoter) targets associated with various cancers are described above and shown below in Table 4. It will be recognized that methylation (forward strand and/or reverse strand) of one or more of the genes shown in Table 4 for each cancer can be determined to identify, and/or stage, and/or provide a prognosis for the indicated cancer. In certain embodiments methylation status of all of the genes shown for a particular cancer (forward and/or reverse strand) can be determined in a single multiplex PCR reaction. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Illustrative primers and probes for the detection of methylation 
               
               
                 at the promoters of genes associated with various cancers 
               
               
                 using the devices and methods described herein. 
               
               
                 Primer and probe numbers refer to primer/probe numbers 
               
               
                 (primer/probe num) shown in Table 5, below. 
               
            
           
           
               
               
               
               
            
               
                 Indication/ 
                 External 
                 Internal 
                   
               
               
                 Gene 
                 Primers 
                 Primers 
                 Probe 
               
               
                   
               
            
           
           
               
            
               
                 Breast Cancer 
               
            
           
           
               
               
               
               
            
               
                 AKR1B1: 
                 58/183 
                 19/20 
                 193 
               
               
                 HIST1H3C: 
                 42/43, 
                 59/54 
                 194 
               
               
                 HOXB4: 
                 186/187, 
                 25/26 
                 76 
               
               
                 RASGRF2: 
                 188/199, 
                 192/14  
                 67 
               
               
                 RASSF1A: 
                 189/1,   
                 1/2 
                 63 
               
               
                 TM6SF1: 
                 202/51,  
                 31/57 
                 77 
               
               
                 BG: 
                 175/158, 
                 176/156 
                 164 
               
            
           
           
               
            
               
                 Pancreatic Cancer Set 1: 
               
            
           
           
               
               
               
               
            
               
                 BNC1 
                 213/214, 
                 221/222 
                 229 
               
               
                 ADAMTS1: 
                 219/220, 
                 227/228 
                 265 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
            
           
           
               
            
               
                 Pancreatic Cancer Set 2: 
               
            
           
           
               
               
               
               
            
               
                 BNC1: 
                 217/218, 
                 225/226 
                 264 
               
               
                 ADAMTS1: 
                 215/216, 
                 223/224 
                 230 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
            
           
           
               
            
               
                 Lung CPHD Set 2: 
               
            
           
           
               
               
               
               
            
               
                 CDO1: 
                 283/284, 
                 287/288 
                 291 
               
               
                 TAC1: 
                 293/294, 
                 386/388 
                 301 
               
               
                 SOX17: 
                 303/304, 
                 382/385 
                 312 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
            
           
           
               
            
               
                 Prostate: 
               
            
           
           
               
               
               
               
            
               
                 GSTP1: 
                 233/234, 
                 239/240 
                 245 
               
               
                 APC: 
                 235/236, 
                 241/242 
                 246 
               
               
                 PTGS2: 
                 237/238, 
                 243/244 
                 247 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
            
           
           
               
            
               
                 BRCA1: 
               
            
           
           
               
               
               
               
            
               
                 BRCA1: 
                 328/329, 
                 330/331 
                 327 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
            
           
           
               
            
               
                 MGMT: 
               
            
           
           
               
               
               
               
            
               
                 MGMT: 
                 248b/249b, 
                 250/251 
                 252 
               
               
                 ACTB: 
                 102/103, 
                 320/321 
                 150 
               
               
                   
               
            
           
         
       
     
     Illustrative primers and probes for the detection of methylation at the promoters of various genes are shown below in Table 5, below, and in Tables 11 and 12 in Example 4. In certain embodiments these primers and/or probes are particularly suitable for use in a multiplex amplification. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Illustrative primers and probes for the detection of methylation of 
               
               
                 various gene promoters. Note Y is C/T; R is A/G; C* is an optionally 
               
               
                 functionalized (e.g., to alter probe T m ) C; T* is an optionally 
               
               
                  functionalized (e.g., to alter probe T m ) T; A* is an optionally 
               
               
                 functionalized (e.g., to alter probe T m ) A; N* is a nucleotide 
               
               
                 optionally a quencher; dP is a universal pyrimidine; 
               
               
                  dK is a universal purine. 
               
            
           
           
               
               
               
               
               
            
               
                 Primer/ 
                   
                   
                   
                 SEQ 
               
               
                 Probe 
                   
                   
                   
                 ID 
               
               
                 Num 
                 Target 
                 Type 
                 Sequence 
                 NO 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   1 
                 RASSF1A 
                 I 
                 GCGTTGAAGTCGGGGTTC 
                 2 
               
               
                   
               
               
                   2 
                 RASSF1A 
                 I 
                 CCCGTACTTCGCTAACTTTAAACG 
                 3 
               
               
                   
               
               
                   3 
                 RASSF1A 
                 P 
                 ACAAACGCGAACCGAACGAAACCA- 
                 4 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                   4 
                 RASSF1A STD 
                 I 
                 fluor-TTAGGGTAGATTGTGGATATTAG 
                 5 
               
               
                   
               
               
                   5 
                 RASSF1A STD 
                 I 
                 ATACTAACAACTATCCAATACAAC 
                 6 
               
               
                   
               
               
                   6 
                 RASSF1A STD 
                 P 
                 fluor-(C*)AGGTTGAAATTAG(T- 
                 7 
               
               
                   
                   
                   
                 quencher)ATGTGTTATTTTGGTATGG 
                   
               
               
                   
               
               
                   7 
                 HIST1H3C 
                 I 
                 AATAGTTCGTAAGTTTATCGGCG 
                 8 
               
               
                   
               
               
                   8 
                 HIST1H3C 
                 I 
                 CTTCACGCCACCGATAACCGA 
                 9 
               
               
                   
               
               
                   9  
                 HIST1H3C 
                 P 
                 fluor- 
                 10 
               
               
                   
                   
                   
                 TACTTACGCGAAACTTTACCGCCGA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  10 
                 HIST1H3C STD 
                 I 
                 GATTTAGAGTTGGATGTGTGGAT 
                 11 
               
               
                   
               
               
                  11 
                 HIST1H3C STD 
                 I 
                 ACCACCATACTAATAATCAAATCTA 
                 12 
               
               
                   
               
               
                  12 
                 HIST1H3C STD 
                 P 
                 fluor- 
                 13 
               
               
                   
                   
                   
                 AAATATCACTCATCACCAAATAAATCCA 
                   
               
               
                   
                   
                   
                 A-quencher/blocker 
                   
               
               
                   
               
               
                  13 
                 RASGRF2 
                 I 
                 GTAAGAAGACGGTCGAGGCG 
                 14 
               
               
                   
               
               
                  14 
                 RASGRF2 
                 I 
                 ACAACTCTACTCGCCCTCGAA 
                 15 
               
               
                   
               
               
                  15 
                 RASGRF2 
                 P 
                 fluor- 
                 16 
               
               
                   
                   
                   
                 AACGAACCACTTCTCGTACCAACGA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  16 
                 RASGRF2 STD 
                 I 
                 TGTATGAGTTTGTGGTGAATAATG 
                 17 
               
               
                   
               
               
                  17 
                 RASGRF2 STD 
                 I 
                 AACTCACCATCAAACACTTTCCC 
                 18 
               
               
                   
               
               
                  18 
                 RASGRF2 STD 
                 P 
                 fluor- 
                 19 
               
               
                   
                   
                   
                 TACAAACCCAACATCCTCTATCTATTC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  19 
                 AKR1B1 
                 I 
                 GCGCGTTAATCGTAGGCGTTT 
                 20 
               
               
                   
               
               
                  20 
                 AKR1B1 
                 I 
                 CCCAATACGATACGACCTTAAC 
                 21 
               
               
                   
               
               
                  21 
                 AKR1B1 
                 P 
                 fluor- 
                 22 
               
               
                   
                   
                   
                 CGTACCTTTAAATAACCCGTAAAATCGA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  22 
                 AKR1B1 STD 
                 I 
                 TTTGTTGATGTTTTGTGGAAGTAAG 
                 23 
               
               
                   
               
               
                  23 
                 AKR1B1 STD 
                 I 
                 ATTCATCAATACTTTCAAATAACACA 
                 24 
               
               
                   
               
               
                  24 
                 AKR1B1 STD 
                 P 
                 fluor-(C*)AAATACATTATCC(T- 
                 25 
               
               
                   
                   
                   
                 quencher)ACCACTAACAATACA 
                   
               
               
                   
               
               
                  25 
                 HOXB4 
                 I 
                 CGGGATTTTGGGTTTTCGTCG 
                 26 
               
               
                   
               
               
                  26 
                 HOXB4 
                 I 
                 CGACGAATAACGACGCAAAAAC 
                 27 
               
               
                   
               
               
                  27 
                 HOXB4 
                 P 
                 fluor- 
                 28 
               
               
                   
                   
                   
                 AACCGAACGATAACGAAAACGACGAA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  28 
                 HOXB4 STD 
                 I 
                 GTTAGTTTTGTAGTGTATTGAGTAT 
                 29 
               
               
                   
               
               
                  29 
                 HOXB4 STD 
                 I 
                 CATCTTCCACAATAAACTTCCAATT 
                 30 
               
               
                   
               
               
                  30 
                 HOXB4 STD 
                 P 
                 fluor- 
                 31 
               
               
                   
                   
                   
                 TAACTCCACCTATTCTACCTACCATTT- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  31 
                 TM6SF1 
                 I 
                 CGTTTAGCGGGATGCGGTGA 
                 32 
               
               
                   
               
               
                  32 
                 TM6SF1 
                 I 
                 ACACGAAAACCCCGATAACCG 
                 33 
               
               
                   
               
               
                  33 
                 TM6SF1 
                 P 
                 fluor-AAACACTCATCGCAACCGCCGCG- 
                 34 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  34 
                 TM6SF1 STD 
                 I 
                 TTAGATGTTGATTGGTTGTGTTTG 
                 35 
               
               
                   
               
               
                  35 
                 TM6SF1 STD 
                 I 
                 ATCATCATAAAACTCAACAATCAATT 
                 36 
               
               
                   
               
               
                  36 
                 TM6SF1 STD 
                 P 
                 fluor- 
                 37 
               
               
                   
                   
                   
                 CCAAACATCAAATCTTTAACTTTTACCA 
                   
               
               
                   
                   
                   
                 A-quencher/blocker 
                   
               
               
                   
               
               
                  37 
                 RASSF1A STD 
                 P 
                 fluor-AGGTTGAAATTAGTATG(T- 
                 38 
               
               
                   
                   
                   
                 quencher)GTTATTTTGGTATGG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  38 
                 RASSF1A STD 
                 P 
                 fluor-AGGTTGAAATTAGTATGTGT(T- 
                 39 
               
               
                   
                   
                   
                 quencher)ATTTTGGTATGG-quencher/blocker 
                   
               
               
                   
               
               
                  39 
                 RASSF1A STD 
                 P 
                 fluor-AGGTTGAAATTAGTATGTGTTA(T- 
                 40 
               
               
                   
                   
                   
                 quencher)TTTGGTATGG-quencher/blocker 
                   
               
               
                   
               
               
                  40 
                 RASSF1A 
                 E 
                 GTTTTATAGTTTTTGTATTTAGG 
                 41 
               
               
                   
               
               
                  41 
                 RASSF1A 
                 E 
                 AACTCAATAAACTCAAACTCCC 
                 42 
               
               
                   
               
               
                  42 
                 HIST1H3C 
                 E 
                 GTGTGTGTTTTTATTGTAAATGG 
                 43 
               
               
                   
               
               
                  43 
                 HIST1H3C 
                 E 
                 ATAAAATTTCTTCACRCCACC 
                 44 
               
               
                   
               
               
                  44 
                 RASGRF2 
                 E 
                 GAGGGAGTTAGTTGGGTTAT 
                 45 
               
               
                   
               
               
                  45 
                 RASGRF2 
                 E 
                 CCTCCAAAAAATACATACCC 
                 46 
               
               
                   
               
               
                  46 
                 AKR1B1 
                 E 
                 GTGTAATTAATTAGAAGGTTTTTT 
                 47 
               
               
                   
               
               
                  47 
                 AKR1B1 
                 E 
                 AACACCTACCTTCCAAATAC 
                 48 
               
               
                   
               
               
                  48 
                 HOXB4 
                 E 
                 TTAGAGGYGAGAGAGTAGTT 
                 49 
               
               
                   
               
               
                  49 
                 HOXB4 
                 E 
                 AAACTACTACTAACCRCCTC 
                 50 
               
               
                   
               
               
                  50 
                 TM6SF1 
                 E 
                 AGGAGATATYGTTGAGGGGA 
                 51 
               
               
                   
               
               
                  51 
                 TM6SF1 
                 E 
                 TCACTCATACTAAACCRCCAA 
                 52 
               
               
                   
               
               
                  52 
                 RASSF1A STD 
                 I 
                 TTAGGGTAGATTGTGGATATTAGATAGG 
                 53 
               
               
                   
               
               
                  53 
                 RASSF1A STD 
                 I 
                 TAATACTAACAACTATCCAATACAACAC 
                 54 
               
               
                   
               
               
                  54 
                 HIST1H3C 
                 I 
                 CCGATAACCGAAACGCTCTTAC 
                 55 
               
               
                   
               
               
                  55 
                 AKR1B1 
                 I 
                 GCGTTAATCGTAGGCGTTT 
                 56 
               
               
                   
               
               
                  56 
                 TM6SF1 
                 I 
                 GTTTAGCGGGATGCGGTG 
                 57 
               
               
                   
               
               
                  57 
                 TM6SF1 
                 I 
                 ACACGAAAACCCCGATAAC 
                 58 
               
               
                   
               
               
                  58 
                 AKR1B1 
                 E 
                 GYGTAATTAAT(T*)AGAAGGTTTTTT 
                 59 
               
               
                   
               
               
                  59 
                 HIST1H3C 
                 I 
                 TCGTACGAAGTAAATAGTTCGTAAG 
                 60 
               
               
                   
               
               
                  60 
                 HIST1H3C 
                 E 
                 GGATTTTTGAAATATTATAGGATTAATT 
                 61 
               
               
                   
                   
                   
                 AG 
                   
               
               
                   
               
               
                  61 
                 RASSF1A 
                 E 
                 GTTTTATAGTT(T*)TTGTATTTAGG 
                 62 
               
               
                   
               
               
                  62 
                 RASSF1A 
                 P 
                 fluor- 
                 63 
               
               
                   
                   
                   
                 ACAAACGCGA(N*)ACCGAA(C**)GAAAC 
                   
               
               
                   
                   
                   
                 CA-quencher/blocker 
                   
               
               
                   
               
               
                  63 
                 RASSF1A 
                 P 
                 fluor-(C*)TGGTTTCGT(T- 
                 64 
               
               
                   
                   
                   
                 quencher)CGGT(T*)CGCG-quencher/blocker 
                   
               
               
                   
               
               
                  64 
                 RASSF1A STD 
                 P 
                 fluor-(C*)AGGTTGAAATTAGTA(T- 
                 65 
               
               
                   
                   
                   
                 quencher)GTGTTAT(T*)TTGG(T*)ATGG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  65 
                 HIST1H3C 
                 P 
                 fluor- 
                 66 
               
               
                   
                   
                   
                 CAAACTACTTACGCGAAACTT(T*)ACCG 
                   
               
               
                   
                   
                   
                 CC-quencher/blocker 
                   
               
               
                   
               
               
                  66 
                 HIST1H3C STD 
                 P 
                 fluor- 
                 67 
               
               
                   
                   
                   
                 AAATATCACTCA(T*)CACCAAA(N*)TAA 
                   
               
               
                   
                   
                   
                 A(T*)CCAA-quencher/blocker 
                   
               
               
                   
               
               
                  67 
                 RASGRF2 
                 P 
                 fluor- 
                 68 
               
               
                   
                   
                   
                 AAACGAACCACTTCTCG(T*)ACCAACGA 
                   
               
               
                   
                   
                   
                 C-quencher/blocker 
                   
               
               
                   
               
               
                  68 
                 RASGRF2 STD 
                 P 
                 fluor- 
                 69 
               
               
                   
                   
                   
                 CAAACCCAACATCCTC(T*)ATC(T*)ATTC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  69 
                 AKR1B1 
                 P 
                 fluor- 
                 70 
               
               
                   
                   
                   
                 A(C*)GCGTACCTTT(N*)AAA(T*)AACCCG 
                   
               
               
                   
                   
                   
                 (T*)AAAATCG-quencher/blocker 
                   
               
               
                   
               
               
                  70 
                 AKR1B1 
                 P 
                 fluor-A(C*)GCGTACCTT(T- 
                 71 
               
               
                   
                   
                   
                 quencher)AAA(T*)AACCCG(T*)AAAATCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  71 
                 AKR1B1 STD 
                 P 
                 fluor-(C*)AAATACATTATCC(T- 
                 72 
               
               
                   
                   
                   
                 quencher)ACCAC(T*)AACAA(T*)ACA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  72 
                 HOXB4 
                 P 
                 fluor- 
                 73 
               
               
                   
                   
                   
                 AACCGAACGATAACGAAAA(C**)GACGA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  73 
                 HOXB4 STD 
                 P 
                 fluor- 
                 74 
               
               
                   
                   
                   
                 TAACTCCACCTATTC(T*)ACCT(N*)ACCA 
                   
               
               
                   
                   
                   
                 (T*)TT-quencher/blocker 
                   
               
               
                   
               
               
                  74 
                 TM6SF1 STD 
                 P 
                 fluor- 
                 75 
               
               
                   
                   
                   
                 CAAACATCAAATCT(T*)TAAC(T*)TT(T*) 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                  75 
                 AKR1B1 
                 P 
                 fluor-(C*)A(C*)GCGTACCT(T- 
                 76 
               
               
                   
                   
                   
                 quencher)TAAA(T*)AACCCG(T*)AAAATCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  76 
                 HOXB4 
                 P 
                 fluor- 
                 77 
               
               
                   
                   
                   
                 AACCGAACGA(T*)AACGAAA(N*)ACGAC 
                   
               
               
                   
                   
                   
                 GAA-quencher/blocker 
                   
               
               
                   
               
               
                  77 
                 TM6SF1 
                 P 
                 fluor-AAACACTCATCGCAACCGCCGCG- 
                 78 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  78 
                 ACTB 
                 P 
                 fluor- 
                 79 
               
               
                   
                   
                   
                 TAACCACCACCCAACACA(C**)AATAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  79 
                 ALU Long Set 1 
                 P 
                 fluor- 
                 80 
               
               
                   
                   
                   
                 CCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  80 
                 ALU Short Set 1 
                 P 
                 fluor- 
                 81 
               
               
                   
                   
                   
                 CACCTAAAA(T*)CAAAAATT(T*)AAAAC 
                   
               
               
                   
                   
                   
                 C-quencher/blocker 
                   
               
               
                   
               
               
                  81 
                 ALU Long Set 2 
                 P 
                 fluor- 
                 82 
               
               
                   
                   
                   
                 CAAATAATTCTCC(T*)ACCTCAACC(T*)C- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  82 
                 ALU Short Set 2 
                 P 
                 fluor- 
                 83 
               
               
                   
                   
                   
                 CTTAACTCAC(T*)ACAACCTC(T*)ACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  83 
                 INSL6 
                 P 
                 fluor- 
                 84 
               
               
                   
                   
                   
                 CAAACCGAACGACGCGCACAAACAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                  84 
                 ACTB 
                 E 
                 GTATATAGGTTGGGGAAGTTTG 
                 85 
               
               
                   
               
               
                  85 
                 ACTB 
                 E 
                 AACTATACTCAACCAATAAAACC 
                 86 
               
               
                   
               
               
                  86 
                 ALU Long Set 1 
                 E 
                 TGTTATTTAGGTTGGAGTGTAG 
                 87 
               
               
                   
               
               
                  87 
                 ALU Long Set 1 
                 E 
                 TAATAACTCATACCTATAATCCC 
                 88 
               
               
                   
               
               
                  88 
                 ALU Long Set 1 
                 I 
                 GGTTGGAGTGTAGTGGTATAATTTTAG 
                 89 
               
               
                   
               
               
                  89 
                 ALU Long Set 1 
                 I 
                 TAATAACTCATACCTATAATCCCAACAC 
                 90 
               
               
                   
               
               
                  90 
                 ALU Short Set 1 
                 E 
                 GTAGAGATAGGGTTTTATTATGTTG 
                 91 
               
               
                   
               
               
                  91 
                 ALU Short Set 1 
                 I 
                 GGTTTTATTATGTTGGTTAGGTTGG 
                 92 
               
               
                   
               
               
                  92 
                 ALU Long Set 2 
                 E 
                 GTATTTTGGGAGGTTAAGGTAG 
                 93 
               
               
                   
               
               
                  93 
                 ALU Long Set 2 
                 E 
                 ATCTTACTCTTATTACCCAAAC 
                 94 
               
               
                   
               
               
                  94 
                 ALU Long Set 2 
                 I 
                 GGTTAAGGTAGGTAGATTATTTGAGG 
                 95 
               
               
                   
               
               
                  95 
                 ALU Long Set 2 
                 I 
                 ATCTTACTCTTATTACCCAAACTAAAATA 
                 96 
               
               
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                  96 
                 ALU Short Set 2 
                 E 
                 GTTATTTAGGAGGTTGAGGTAG 
                 97 
               
               
                   
               
               
                  97 
                 ALU Short Set 2 
                 E 
                 GAGGTAGGAGAATTATTTGAATTTAGG 
                 98 
               
               
                   
               
               
                  98 
                 INSL6 
                 E 
                 ATTTGAGATTTTTGAGTTGG 
                 99 
               
               
                   
               
               
                  99 
                 INSL6 
                 E 
                 AACCCTACTCCCTATCTACG 
                 100 
               
               
                   
               
               
                 100 
                 INSL6 
                 I 
                 GCGCGCGTTTTTTTTTGAAG 
                 101 
               
               
                   
               
               
                 101 
                 INSL6 
                 I 
                 GGCGTAGATAGGGAGTAGGGTT 
                 102 
               
               
                   
               
               
                 102 
                 ACTB 
                 I 
                 GTGATGGAGGAGGTTTAGTAAGTT 
                 103 
               
               
                   
               
               
                 103 
                 ACTB 
                 I 
                 CCAATAAAACCTACTCCTCCCTTAA 
                 104 
               
               
                   
               
               
                 104 
                 RASSF1A STD 
                 P 
                 fluor-(C*)(C*)ATACCAAAA(T- 
                 105 
               
               
                   
                   
                   
                 quencher)AACACA(T*)CTAAT(T*)TCAACC 
                   
               
               
                   
                   
                   
                 T-quencher/blocker 
                   
               
               
                   
               
               
                 105 
                 AKR1B1 STD 
                 P 
                 fluor-(C*)AAATACAT(T*)ATCC(T- 
                 106 
               
               
                   
                   
                   
                 quencher)ACCAC(T*)AACAA(T*)ACA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 106 
                 AKR1B1 
                 P 
                 fluor-(C*)ACGCGTACCTT(T- 
                 107 
               
               
                   
                   
                   
                 quencher)AAA(T*)AACCCG(T*)AAAATCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 107 
                 AKR1B1 
                 P 
                 fluor-(C*)ACGCGTACCTT(T*)AAA(T- 
                 108 
               
               
                   
                   
                   
                 quencher)AACCCG(T*)AAAATCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 108 
                 RASSF1A UM 
                 P 
                 fluor-CTAACAAACA(C- 
                 109 
               
               
                   
                   
                   
                 quencher)AAA(C**)CAAA(C**)AAAACCA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 109 
                 RASSF1A UM 
                 P 
                 fluor-CTAACAAACA(C**)AAA(C- 
                 110 
               
               
                   
                   
                   
                 quencher)CAAA(C**)AAAACCA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 110 
                 HIST1H3C UM 
                 P 
                 fluor- 
                 111 
               
               
                   
                   
                   
                 AACTACTTACA(C**)AAAACTT(N*)TAC(C**) 
                   
               
               
                   
                   
                   
                 ACCAA-quencher/blocker 
                   
               
               
                   
               
               
                 111 
                 HIST1H3C UM 
                 P 
                 fluor- 
                 112 
               
               
                   
                   
                   
                 AACTACTTA(C**)ACAAAA(C**)TTTACC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 112 
                 RASGRF2 UM 
                 P 
                 fluor- 
                 113 
               
               
                   
                   
                   
                 AAACAAACCAC(T*)TCTCA(T*)ACCAACA 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 113 
                 AKR1B1 UM 
                 P 
                 fluor-(C*)ACATACCTTTAAA(T- 
                 114 
               
               
                   
                   
                   
                 quencher)AACCCA(T*)AAAA(T*)CAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 114 
                 AKR1B1 UM 
                 P 
                 fluor-(C*)ACATACCTT(T- 
                 115 
               
               
                   
                   
                   
                 quencher)AAA(T*)AACCCA(T*)AAAATCA 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 115 
                 HOXB4 UM 
                 P 
                 fluor- 
                 116 
               
               
                   
                   
                   
                 CAACAAAAACCCAAAA(T*)CCCAAC(N*) 
                   
               
               
                   
                   
                   
                 AAACCACA-quencher/blocker 
                   
               
               
                   
               
               
                 116 
                 HOXB4 UM 
                 P 
                 fluor- 
                 117 
               
               
                   
                   
                   
                 CAAAATCCCAA(C**)AAACCA(C**)ATAA 
                   
               
               
                   
                   
                   
                 CA-quencher/blocker 
                   
               
               
                   
               
               
                 117 
                 TM6SF1 UM 
                 P 
                 fluor- 
                 118 
               
               
                   
                   
                   
                 AAACACTCATCACAACCA(C**)CACACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 118 
                 AKR1B1 UM 
                 I 
                 TGGTGTGTTAATTGTAGGTGTTTT 
                 119 
               
               
                   
               
               
                 119 
                 AKR1B1 UM 
                 I 
                 CCCAATACAATACAACCTTAACC 
                 120 
               
               
                   
               
               
                 120 
                 HOXB4 UM 
                 I 
                 GTGGTGTGTATTGTGTAGTGTTA 
                 121 
               
               
                   
               
               
                 121 
                 HOXB4 UM 
                 I 
                 CAAACCAAACAATAACAAAAACAAC 
                 122 
               
               
                   
               
               
                 122 
                 TM6SF1 UM 
                 I 
                 TGTTTAGTGGGATGTGGTGAAG 
                 123 
               
               
                   
               
               
                 123 
                 TM6SF1 UM 
                 I 
                 ACACAAAAACCCCAATAACCACA 
                 124 
               
               
                   
               
               
                 124 
                 RASSF1A UM 
                 I 
                 GTTTAAAGTTAGTGAAGTATGGGTTT 
                 125 
               
               
                   
               
               
                 125 
                 HIST1H3C UM 
                 I 
                 TGTATGAAGTAAATAGTTTGTAAGTTTA 
                 126 
               
               
                   
                   
                   
                 TTGG 
                   
               
               
                   
               
               
                 126 
                 AKR1B1 STD 
                 I 
                 TTTGTTGATGTTTTGTGGAAG(T*)AAG 
                 127 
               
               
                   
               
               
                 127 
                 AKR1B1 STD 
                 I 
                 ATTCATCAATACTTTCAAA(T*)AACACA 
                 128 
               
               
                   
               
               
                 128 
                 RASGRF2 
                 P 
                 fluor- 
                 129 
               
               
                   
                   
                   
                 AAACGAACCACTTCTCG(T*)ACCAACGA 
                   
               
               
                   
                   
                   
                 C-quencher/blocker 
                   
               
               
                   
               
               
                 129 
                 RASGRF2 STD 
                 P 
                 fluor- 
                 130 
               
               
                   
                   
                   
                 CAAACCCAACATCCTC(T*)ATC(T*)ATTC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 130 
                 TM6SF1 
                 P 
                 fluor-AAACACTCATCGCAACCGCCGCG- 
                 131 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 131 
                 TM6SF1 STD 
                 P 
                 fluor- 
                 132 
               
               
                   
                   
                   
                 CCAAACATCAAATCT(T*)TAACTT(T*)TA 
                   
               
               
                   
                   
                   
                 CCAA-quencher/blocker 
                   
               
               
                   
               
               
                 132 
                 TM6SF1 
                 P 
                 fluor-AAACACTCATCGCAACCGCCGCG- 
                 133 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 133 
                 RASSF1A UM 
                 I 
                 GGTGTTGAAGTTGGGGTTTG 
                 134 
               
               
                   
               
               
                 134 
                 RASSF1A UM 
                 I 
                 CCCATACTTCACTAACTTTAAAC 
                 135 
               
               
                   
               
               
                 135 
                 HIST1H3C UM 
                 I 
                 GTAAATAGTTTGTAAGTTTATTGGTG 
                 136 
               
               
                   
               
               
                 136 
                 HIST1H3C UM 
                 I 
                 TTTCTTCACACCACCAATAACCAA 
                 137 
               
               
                   
               
               
                 137 
                 RASGRF2 UM 
                 I 
                 GAGTAAGAAGATGGTTGAGGTG 
                 138 
               
               
                   
               
               
                 138 
                 RASGRF2 UM 
                 I 
                 CAACAACTCTACTCACCCTCAA 
                 139 
               
               
                   
               
               
                 139 
                   
                 P 
                 fluor- 
                 140 
               
               
                   
                   
                   
                 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 140 
                 ALU Long Set 1 
                 P 
                 fluor- 
                 141 
               
               
                   
                   
                   
                 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 141 
                 ALU Long Set 1 
                 P 
                 fluor- 
                 142 
               
               
                   
                   
                   
                 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 142 
                 ALU Long Set 1 
                 P 
                 fluor- 
                 143 
               
               
                   
                   
                   
                 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 143 
                 ALU Long Set 1 
                 P 
                 fluor- 
                 144 
               
               
                   
                   
                   
                 TCCCAACTACT(T*)AAAAAAC(T*)AAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 144 
                 HMBS 
                 I 
                 GGATAAGATTTTTGATATTGTATTTTTTA 
                 145 
               
               
                   
                   
                   
                 AGG 
                   
               
               
                   
               
               
                 145 
                 HMBS 
                 I 
                 CATATTCAAACTCCTTAATAAACAAACT 
                 146 
               
               
                   
                   
                   
                 TTTCTC 
                   
               
               
                   
               
               
                 146 
                 HMBS 
                 P 
                 fluor-CCGAACAAAAAAAA(C- 
                 147 
               
               
                   
                   
                   
                 quencher)CTAAA(T*)AAATCCC(T*)TC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 147 
                 ACTB 
                 P 
                 fluor- 
                 148 
               
               
                   
                   
                   
                 CCACCACCCAACACACAA(T*)AACAAAC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 148 
                 ACTB 
                 I 
                 GGTTTAGTAAGTTTTTTGGATTGTG 
                 149 
               
               
                   
               
               
                 149 
                 ACTB 
                   
                 CCTTAAAAATTACAAAAACCACAAC 
                 150 
               
               
                   
               
               
                 150 
                 ACTB 
                 P 
                 fluor-CCACCACCCAACA(C- 
                 151 
               
               
                   
                   
                   
                 quencher)ACAA(T*)AACAAACAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 151 
                 ACTB 
                 P 
                 fluor- 
                 152 
               
               
                   
                   
                   
                 CCACCACCCAAC(N*)ACA(C**)AATAA(C**) 
                   
               
               
                   
                   
                   
                 AAACAC-quencher/blocker 
                   
               
               
                   
               
               
                 152 
                 ACTB 
                 P 
                 fluor- 
                 153 
               
               
                   
                   
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACA 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 153 
                 BG 
                 I 
                 TTCAGTGCCGGTTGGTAATGTAA- 
                 154 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 154 
                 BG 
                 I 
                 CAACAACTTTAATACCTGTTTCAAGGA 
                 155 
               
               
                   
               
               
                 155 
                 BG conv 
                 I 
                 GGTATTTTTGTATTTGTTGGTGTTG 
                 156 
               
               
                   
               
               
                 156 
                 BG conv 
                 I 
                 CATACATACACCAAACAATTCATTC 
                 157 
               
               
                   
               
               
                 157 
                 BG conv 
                 E 
                 GTATGGTGGTATTTTTGTATTTGTTG 
                 158 
               
               
                   
               
               
                 158 
                 BG conv 
                 E 
                 CACACATACATACACCAAACAATTC 
                 159 
               
               
                   
               
               
                 159 
                 BG 
                 P 
                 fluor- 
                 160 
               
               
                   
                   
                   
                 AAGATCCGATTCACAGA(N*)CAAGCTCC 
                   
               
               
                   
                   
                   
                 GTCA-quencher/blocker 
                   
               
               
                   
               
               
                 160 
                 BG 
                 I 
                 fluor- 
                 161 
               
               
                   
                   
                   
                 AAGATCCGATTCACAGA(N*)CAAGCTCC 
                   
               
               
                   
                   
                   
                 GTCA-quencher/blocker 
                   
               
               
                   
               
               
                 161 
                 BG conv 
                 P 
                 fluor-(C*)AAAATCATTT(C- 
                 162 
               
               
                   
                   
                   
                 quencher)CTT(C**)ACAAATA(C**)ACTC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 162 
                 BG conv 
                 P 
                 fluor-CCAAATACCA(T- 
                 163 
               
               
                   
                   
                   
                 quencher)AACCA(T*)TTTATTAA(T*)AACA 
                   
               
               
                   
                   
                   
                 C-quencher/blocker 
                   
               
               
                   
               
               
                 163 
                 BG conv 
                 P 
                 fluor- 
                 164 
               
               
                   
                   
                   
                 AAAATCATTTCCTT(C**)ACA(N*)AATA(C**) 
                   
               
               
                   
                   
                   
                 ACTC-quencher/blocker 
                   
               
               
                   
               
               
                 164 
                 BG conv 
                 P 
                 fluor- 
                 165 
               
               
                   
                   
                   
                 CCAAATACCA(T*)AACCAT(N*)TTTATTA 
                   
               
               
                   
                   
                   
                 A(T*)AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 165 
                 short HMBS 
                 I 
                 CCCTAGTATGCTAGGTCTCTTGCTGGGA 
                 166 
               
               
                   
               
               
                 166 
                 short HMBS 
                 I 
                 CAGCCTCTCTGAGGGTTTAAGCCCA 
                 167 
               
               
                   
               
               
                 167 
                 short HMBS 
                 P 
                 fluor- 
                 168 
               
               
                   
                   
                   
                 TCAGCC(T*)ATC(T*)GACACCCCGGG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 168 
                 short β-Globin 
                 I 
                 GACTCCTGAGGAGAAGTCTGCCGTTA 
                 169 
               
               
                   
               
               
                 169 
                 short β-Globin 
                 I 
                 CCTTGATACCAACCTGCCCAGGG 
                 170 
               
               
                   
               
               
                 170 
                 short β-Globin 
                 P 
                 fluor- 
                 171 
               
               
                   
                   
                   
                 AGGTGAACG(T*)GGATGAAGT(T*)GGTG 
                   
               
               
                   
                   
                   
                 GTG-quencher/blocker 
                   
               
               
                   
               
               
                 171 
                 short BG 
                 I 
                 CAACATCGCGCAAGAGCACGG 
                 172 
               
               
                   
               
               
                 172 
                 short BG 
                 I 
                 CGTTTCCTTCACGAGTACGCTCTCCGA 
                 173 
               
               
                   
               
               
                 173 
                 short BG 
                 P 
                 fluor- 
                 174 
               
               
                   
                   
                   
                 ACCGGCGAA(T*)ACAGAGA(T*)ACCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 174 
                 ACTB 
                 P 
                 fluor- 
                 175 
               
               
                   
                   
                   
                 CC(A*)CC(A*)CCCAAC(N*)ACA(C**)AAT 
                   
               
               
                   
                   
                   
                 AA(C**)AAACAC-quencher/blocker 
                   
               
               
                   
               
               
                 175 
                 BG conv 
                 I 
                 GTTGGTGTTGGAGAGTGTATTTG 
                 176 
               
               
                   
               
               
                 176 
                 BG conv 
                 I 
                 GGAGAGTGTATTTGTGAAGGAAATG 
                 177 
               
               
                   
               
               
                 177 
                 BG conv 
                 I 
                 GGAAATGATTTTTTTTATGAGATGAGTG 
                 178 
               
               
                   
               
               
                 178 
                 ACTB 
                 P 
                 fluor- 
                 179 
               
               
                   
                   
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACA 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 179 
                 ACTB 
                 P 
                 fluor- 
                 180 
               
               
                   
                   
                   
                 CCACCACCCAACACA(N*)CAA(T*)AACA 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 180 
                 ACTB 
                 P 
                 fluor- 
                 181 
               
               
                   
                   
                   
                 CCACCACCCAACACACAA(T*)AACAAAC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 181 
                 ACTB 
                 I 
                 GATGGAGGAGGTTTAGTAAGTTTTT 
                 182 
               
               
                   
               
               
                 182 
                 ACTB 
                 I 
                 AATAAAACCTACTCCTCCCTTAAAAA 
                 183 
               
               
                   
               
               
                 183a 
                 AKR1B1 
                 E 
                 CTTACCATAACTACTAC(dK)CTCC 
                 184 
               
               
                   
               
               
                 183b 
                 AKR1B1 
                 E 
                 CTTACCATAACTACTACRCTCC 
                 185 
               
               
                   
               
               
                 184 
                 HIST1H3C 
                 E 
                 GTGTGTGTTTTTATTGTAAATGGT 
                 186 
               
               
                   
               
               
                 185a 
                 HIST1H3C 
                 E 
                 AAC(dK)ATAAC(dK)ATAAAATTTCTTCAC 
                 187 
               
               
                   
               
               
                 185b 
                 HIST1H3C 
                 E 
                 AACRATAACRATAAAATTTCTTCAC 
                 188 
               
               
                   
               
               
                 186a 
                 HOXB4 
                 E 
                 GTTTGT(dP)GGGATTTTGGGT 
                 189 
               
               
                   
               
               
                 186b 
                 HOXB4 
                 E 
                 GTTTGTYGGGATTTTGGGT 
                 190 
               
               
                   
               
               
                 187a 
                 HOXB4 
                 E 
                 CC(dK)AACTCC(dK)AAAAAAAAACC 
                 191 
               
               
                   
               
               
                 187b 
                 HOXB4 
                 E 
                 CCRAACTCCRAAAAAAAAACC 
                 192 
               
               
                   
               
               
                 188a 
                 RASGRF2 
                 E 
                 GGTATTAAG(dP)G(dP)GGTTTTTTG 
                 193 
               
               
                   
               
               
                 188b 
                 RASGRF2 
                 E 
                 GGTATTAAGYGYGGTTTTTTG 
                 194 
               
               
                   
               
               
                 189a 
                 RASSF1A 
                 E 
                 GT(dP)GTTTAGTTTGGATTTTGG 
                 195 
               
               
                   
               
               
                 189b 
                 RASSF1A 
                 E 
                 GTYGTTTAGTTTGGATTTTGG 
                 196 
               
               
                   
               
               
                 190 
                 TM6SF1 
                 E 
                 TTTCGAAGGGTAAGCGTTAAG 
                 197 
               
               
                   
               
               
                 191a 
                 TM6SF1 
                 E 
                 AACATAAATAACC(dK)AAA(T*)AACC 
                 198 
               
               
                   
               
               
                 191b 
                 TM6SF1 
                 E 
                 AACATAAATAACCRAAA(T*)AACC 
                 199 
               
               
                   
               
               
                 192 
                 RASGRF2 
                 I 
                 CGGTTTTTTGAGTAAGAAGACGGTC 
                 200 
               
               
                   
               
               
                 193a 
                 AKR1B1 
                 P 
                 fluor-TACCTTTAAA(T- 
                 201 
               
               
                   
                   
                   
                 quencher)AACCC(dK)(T*)AAAA(T*)CGACA 
                   
               
               
                   
                   
                   
                 A-quencher/blocker 
                   
               
               
                   
               
               
                 193b 
                 AKR1B1 
                 P 
                 fluor-TACCTTTAAA(T- 
                 202 
               
               
                   
                   
                   
                 quencher)AACCCR(T*)AAAA(T*)CGACAA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 194a 
                 HIST1H3C 
                 P 
                 fluor- 
                 203 
               
               
                   
                   
                   
                 ATAACAAACTACT(T*)AC(dK)CGAAAC(T*) 
                   
               
               
                   
                   
                   
                 TTAC-quencher/blocker 
                   
               
               
                   
               
               
                 194b 
                 HIST1H3C 
                 P 
                 fluor- 
                 204 
               
               
                   
                   
                   
                 ATAACAAACTACT(T*)ACRCGAAAC(T*)T 
                   
               
               
                   
                   
                   
                 TAC-quencher/blocker 
                   
               
               
                   
               
               
                 195a 
                 HOXB4 
                 P 
                 fluor- 
                 205 
               
               
                   
                   
                   
                 AACAAACC(dK)AA(C**)GA(T*)AAC(N*)A 
                   
               
               
                   
                   
                   
                 AAAC-quencher/blocker 
                   
               
               
                   
               
               
                 195b 
                 HOXB4 
                 P 
                 fluor- 
                 206 
               
               
                   
                   
                   
                 AACAAACCRAA(C**)GA(T*)AAC(N*)AAA 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 196 
                 RASGRF2 
                 P 
                 fluor- 
                 207 
               
               
                   
                   
                   
                 CACATTCTAA(T*)AAAAAAC(N*)AACCA 
                   
               
               
                   
                   
                   
                 C(T*)TC-quencher/blocker 
                   
               
               
                   
               
               
                 197a 
                 RASSF1A 
                 P 
                 fluor-AACC(dK)AA(C**)GAAA(C- 
                 208 
               
               
                   
                   
                   
                 quencher)CA(C**)AAAAC-quencher/blocker 
                   
               
               
                   
               
               
                 197b 
                 RASSF1A 
                 P 
                 fluor-AACCRAA(C**)GAAA(C- 
                 209 
               
               
                   
                   
                   
                 quencher)CA(C**)AAAAC-quencher/blocker 
                   
               
               
                   
               
               
                 198 
                 TM6SF1 
                 P 
                 fluor- 
                 210 
               
               
                   
                   
                   
                 CAAAAACAC(T*)CATC(N*)CAACC GCC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 199 
                 RASGRF2 
                 E 
                 ACAACCCTCCAAAAAATACATA 
                 211 
               
               
                   
               
               
                 200 
                 BG conv 
                 P 
                 fluor- 
                 212 
               
               
                   
                   
                   
                 CCAAATACCATAACCA(T*)TTTATTAA(T*) 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 201 
                 BG conv 
                 P 
                 fluor- 
                 213 
               
               
                   
                   
                   
                 CCAAATACCATAACCA(T*)TTTATTAA(T*) 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 202a 
                 TM6SF1 
                 E 
                 TTT(dP)GAAGGGTAAG(dP)GTTAAG 
                 214 
               
               
                   
               
               
                 202b 
                 TM6SF1 
                 E 
                 TTTYGAAGGGTAAGYGTTAAG 
                 215 
               
               
                   
               
               
                 203a 
                 TM6SF1 
                 E 
                 CAACAC(dK)AAAACCCC(dK)ATA 
                 216 
               
               
                   
               
               
                 203b 
                 TM6SF1 
                 E 
                 CAACACRAAAACCCCRATA 
                 217 
               
               
                   
               
               
                 204 
                 KRAS Multi 
                 E 
                 CCTGCTGAAAATGACTGAATATAACCGC 
                 218 
               
               
                   
                   
                   
                 TAAGAACCTCTCGGTCAGCTGAT 
                   
               
               
                   
               
               
                 205 
                 KRAS Multi 
                 E 
                 CCTGCTGAAAATGACTGAATATAAAGTC 
                 219 
               
               
                   
                   
                   
                 TCATTATAATCGTTCGAGCTGTT 
                   
               
               
                   
               
               
                 206 
                 KRAS Multi 
                 E 
                 CCTGCTGAAAATGACTGAATATAAGCAG 
                 220 
               
               
                   
                   
                   
                 ACTTGGCGGTAGGTCCGAGCTTG 
                   
               
               
                   
               
               
                 207 
                 KRAS Multi 
                 E 
                 CCTGCTGAAAATGACTGAATATAAGTAT 
                 221 
               
               
                   
                   
                   
                 CCTGAGCACGGTTGCGAGCTGCT 
                   
               
               
                   
               
               
                 208 
                 KRAS Multi 
                 I 
                 CTCTTGCCTACGCC(N*)CCGCTAAGAACC 
                 222 
               
               
                   
                   
                   
                 TCTCGGTC 
                   
               
               
                   
               
               
                 209 
                 KRAS Multi 
                 I 
                 CTCTTGCCTACGCC(N*)AGTCTCATTATA 
                 223 
               
               
                   
                   
                   
                 ATCGTTCG 
                   
               
               
                   
               
               
                 210 
                 KRAS Multi 
                 I 
                 CTCTTGCCTACGCC(N*)GCAGACTTGGCG 
                 224 
               
               
                   
                   
                   
                 GTAGGTCC 
                   
               
               
                   
               
               
                 211 
                 KRAS Multi 
                 I 
                 CTCTTGCCTACGCC(N*)GTATCCTGAGCA 
                 225 
               
               
                   
                   
                   
                 CGGTTGCG 
                   
               
               
                   
               
               
                 212 
                 ACTB 
                 P 
                 fluor- 
                 226 
               
               
                   
                   
                   
                 CCACCACCCAACACACAA(T*)AACAAAC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 213 
                 BNC1 
                 E 
                 CCCRCAAACCRCGAAAACCTC 
                 227 
               
               
                   
               
               
                 214 
                 BNC1 
                 E 
                 GTTTTTTTTYGGGAGAGGTAAATA 
                 228 
               
               
                   
               
               
                 215 
                 ADAMTS1 
                 E 
                 CRCCTCCRAAACTAAAACAAC 
                 229 
               
               
                   
               
               
                 216 
                 ADAMTS1 
                 E 
                 GGGTTATTGTAAAGTTAGGGTG 
                 230 
               
               
                   
               
               
                 217 
                 BNC1 
                 E 
                 GAGGT(dP)GTGGTTTT(dP)GTAGAT 
                 231 
               
               
                   
               
               
                 218 
                 BNC1 
                 E 
                 AAAC(dK)CCAAAAAACTTCAAAAC 
                 232 
               
               
                   
               
               
                 219 
                 ADAMTS1 
                 E 
                 TTTTGTTGGGATAAGAAG(dP)GTTT 
                 233 
               
               
                   
               
               
                 220 
                 ADAMTS1 
                 E 
                 ACCAAAAACTATTACAAAACCAAA 
                 234 
               
               
                   
               
               
                 221 
                 BNC1 
                 I 
                 CCGACGACCGACG 
                 235 
               
               
                   
               
               
                 222 
                 BNC1 
                 I 
                 GGGAGAGGTAAATATCGATAC 
                 236 
               
               
                   
               
               
                 223 
                 ADAMTS1 
                 I 
                 CGCGAAAATTAATACCTAACG 
                 237 
               
               
                   
               
               
                 224 
                 ADAMTS1 
                 I 
                 TTAGGGTGCGTTATCGGAC 
                 238 
               
               
                   
               
               
                 225 
                 BNC1 
                 I 
                 CGGAGGTGTTTGTTTTCGTC 
                 239 
               
               
                   
               
               
                 226 
                 BNC1 
                 I 
                 CGAAAAAAACAAACACCGACACG 
                 240 
               
               
                   
               
               
                 227 
                 ADAMTS1 
                 I 
                 CGTTTTCGGGGTTGAGGTAAC 
                 241 
               
               
                   
               
               
                 228 
                 ADAMTS1 
                 I 
                 CCAAAATACGCTACCGAACGA 
                 242 
               
               
                   
               
               
                 229 
                 BNC1 
                 P 
                 fluor- 
                 243 
               
               
                   
                   
                   
                 AAAAT(A*)TCT(A*)(C**)CCC(C**)(dK)CC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 230 
                 ADAMTS1 
                 P 
                 fluor- 
                 244 
               
               
                   
                   
                   
                 TATTACTCACTCTAC(T*)CAAAAC(T*)CT 
                   
               
               
                   
                   
                   
                 CC-quencher/blocker 
                   
               
               
                   
               
               
                 231 
                 BNC1 
                 P 
                 fluor- 
                 245 
               
               
                   
                   
                   
                 ATATCTTTTACCAACAAA(T*)ACCT(T*)C 
                   
               
               
                   
                   
                   
                 AAA-quencher/blocker 
                   
               
               
                   
               
               
                 232 
                 ADAMTS1 
                 P 
                 fluor- 
                 246 
               
               
                   
                   
                   
                 GTTTT(dP)GTTTTGGTTGCGA(T*)GTTGT- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 233 
                 GSTP1 
                 E 
                 GGGATTTTTTAGAAGAG(dP)GGT 
                 247 
               
               
                   
               
               
                 234 
                 GSTP1 
                 E 
                 TACTCACTAATAAC(dK)AAAAC(T*)AC 
                 248 
               
               
                   
               
               
                 235 
                 APC 
                 E 
                 GGTTTTGTGTTTTATTG(dP)GGAG 
                 249 
               
               
                   
               
               
                 236 
                 APC 
                 E 
                 CCTAAC(dK)AACTACACCAATACAA 
                 250 
               
               
                   
               
               
                 237 
                 PTGS2 
                 E 
                 GAGAGGGGATTTTTTG(dP)GTTT 
                 251 
               
               
                   
               
               
                 238 
                 PTGS2 
                 E 
                 CC(dK)AAAACCAATTCTAAACTAATC 
                 252 
               
               
                   
               
               
                 239 
                 GSTP1 
                 I 
                 TTTTTAGAAGAGCGGTCGGC 
                 253 
               
               
                   
               
               
                 240 
                 GSTP1 
                 I 
                 CTAATAACGAAAACTACGACGACG 
                 254 
               
               
                   
               
               
                 241 
                 APC 
                 I 
                 TTGTGTTTTATTGCGGAGTGC 
                 255 
               
               
                   
               
               
                 242 
                 APC 
                 I 
                 AACCACATATCGATCACGTACG 
                 256 
               
               
                   
               
               
                 243 
                 PTGS2 
                 I 
                 GCGTTTTCGGATTTTAGGGTC 
                 257 
               
               
                   
               
               
                 244 
                 PTGS2 
                 I 
                 AACTAATCGCCTTAAATAAAATACCG 
                 258 
               
               
                   
               
               
                 245 
                 GSTP1 
                 P 
                 fluor- 
                 259 
               
               
                   
                   
                   
                 CCTCC(dK)AACCTTA(T*)AA(N*)AAA(T*) 
                   
               
               
                   
                   
                   
                 AATCCC-quencher/blocker 
                   
               
               
                   
               
               
                 246 
                 APC 
                 P 
                 fluor- 
                 260 
               
               
                   
                   
                   
                 AAAAAC(dK)CCCTAATCC(N*)CA(T*)CCA 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 247 
                 PTGS2 
                 P 
                 fluor- 
                 261 
               
               
                   
                   
                   
                 CACTTAACTTCCTC(T*)CCAAAAATC(T*) 
                   
               
               
                   
                   
                   
                 AAAC-quencher/blocker 
                   
               
               
                   
               
               
                 248a 
                 MGMT 
                 E 
                 GTTTT(T*)AGAA(dP)G(T*)TTTG(dP)GTTT 
                 262 
               
               
                   
               
               
                 248b 
                 MGMT 
                 E 
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT 
                 263 
               
               
                   
               
               
                 249a 
                 MGMT 
                 E 
                 AAAAAAC(T*)CC(dK)CACTCTTCC 
                 264 
               
               
                   
               
               
                 249b 
                 MGMT 
                 E 
                 AAAAAAC(T*)CCRCACTCTTCC 
                 265 
               
               
                   
               
               
                 250 
                 MGMT 
                 I 
                 TTTCGACGTTCGTAGGTTTTCGC 
                 266 
               
               
                   
               
               
                 251 
                 MGMT 
                 I 
                 GCACTCTTCCGAAAACGAAACG 
                 267 
               
               
                   
               
               
                 252a 
                 MGMT 
                 P 
                 fluor- 
                 268 
               
               
                   
                   
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 252b 
                 MGMT 
                 P 
                 fluor- 
                 269 
               
               
                   
                   
                   
                 CCAAACAC(T*)CACCAAATC(N*)CAAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 264 
                 BNC1 
                 P 
                 fluor-ATATCTTTTACCAA(C- 
                 270 
               
               
                   
                   
                   
                 quencher)AAA(T*)ACCT(T*)CAAA- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 265 
                 ADAMTS1 
                 P 
                 fluor- 
                 271 
               
               
                   
                   
                   
                 GTTTT(dP)GTTTTGGTTGCGA(T*)GTTGT- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 283 
                 CDO1 
                 E 
                 GGAGATAA(dP)GGGGTTTTTGG 
                 272 
               
               
                   
               
               
                 284 
                 CDO1 
                 E 
                 CACTAAAAATATACCAAC(dK)ACC 
                 273 
               
               
                   
               
               
                 285 
                 CDO1 
                 E 
                 GGAGAGTTATTTAAGAAAGGTGG 
                 274 
               
               
                   
               
               
                 286 
                 CDO1 
                 E 
                 AAAATTAC(dK)C(dK)AAACCCAC 
                 275 
               
               
                   
               
               
                 287 
                 CDO1 
                 I 
                 CGTGTTCGTAGGGTTTTTTCGTTTTC 
                 276 
               
               
                   
               
               
                 288 
                 CDO1 
                 I 
                 CCAACGACCCTCGAAAAAAAAACG 
                 277 
               
               
                   
               
               
                 289 
                 CDO1 
                 I 
                 GATTTTGCGGGTACGGTTTACGC 
                 278 
               
               
                   
               
               
                 290 
                 CDO1 
                 I 
                 GATCCCTAAAACGCCGAAAACAACG 
                 279 
               
               
                   
               
               
                 291 
                 CDO1 
                 P 
                 fluor-(C*)GTTATTTTT(T- 
                 280 
               
               
                   
                   
                   
                 quencher)TTGGG(T*)GGTT(T*)TTCG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 292 
                 CDO1 
                 P 
                 fluor-C(dK)AAAAACCACC(C- 
                 281 
               
               
                   
                   
                   
                 quencher)AAAAAAAA(T*)AAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 293 
                 TAC1 
                 E 
                 GGATAAATAT(dP)GTAAGGTATTGAG 
                 282 
               
               
                   
               
               
                 294 
                 TAC1 
                 E 
                 CGAAATACTAAATTTCTCTAATTCCTC 
                 283 
               
               
                   
               
               
                 295 
                 TAC1 
                 E 
                 GAGTTTTTTTGGTTTTTT(dP)GAG 
                 284 
               
               
                   
               
               
                 296 
                 TAC1 
                 E 
                 CTAAAATAAATACC(dK)CAAAACAC 
                 285 
               
               
                   
               
               
                 297 
                 TAC1 
                 I 
                 CGCGTTCGGATTTTTTTTTCGGC 
                 286 
               
               
                   
               
               
                 298 
                 TAC1 
                 I 
                 AAATTTCTCTAATTCCTCCGAACGCACG 
                 287 
               
               
                   
               
               
                 299 
                 TAC1 
                 I 
                 GCGTACGTTGGTCGTTTCGTATTTTC 
                 288 
               
               
                   
               
               
                 300 
                 TAC1 
                 I 
                 GCAAAACACTAAACAAACGAAAAAACG 
                 289 
               
               
                   
                   
                   
                 CG 
                   
               
               
                   
               
               
                 301 
                 TAC1 
                 P 
                 fluor- 
                 290 
               
               
                   
                   
                   
                 GTAGTTAT(dP)GAGAG(T*)G(N*)GGAGCG 
                   
               
               
                   
                   
                   
                 A(T*)TAG-quencher/blocker 
                   
               
               
                   
               
               
                 302 
                 TAC1 
                 P 
                 fluor- 
                 291 
               
               
                   
                   
                   
                 CTAATC(dK)CTCCGCAC(T*)CTC(N*)A(T*) 
                   
               
               
                   
                   
                   
                 AACTAC-quencher/blocker 
                   
               
               
                   
               
               
                 303 
                 SOX17 
                 E 
                 GTTTGGAG(dP)GTTATGAGTAG 
                 292 
               
               
                   
               
               
                 304 
                 SOX17 
                 E 
                 CTTCATATCCCC(dK)ATAAAACTC 
                 293 
               
               
                   
               
               
                 305 
                 SOX17 
                 E 
                 GGGTTTTTAGTCGGTTTAGTG 
                 294 
               
               
                   
               
               
                 306 
                 SOX17 
                 E 
                 CTAAAAC(dK)TAAAACTC(dK)AACC 
                 295 
               
               
                   
               
               
                 307 
                 SOX17 
                 I 
                 GATTTAGAGCGCGTTGTTCGC 
                 296 
               
               
                   
               
               
                 308 
                 SOX17 
                 I 
                 CATATCCCCGATAAAACTCAACGACTCG 
                 297 
               
               
                   
               
               
                 309 
                 SOX17 
                 I 
                 GTCGGTTTAGTGATATTGCGGGC 
                 298 
               
               
                   
               
               
                 310 
                 SOX17 
                 I 
                 CCACGACCTAAACGTAAACCTAACG 
                 299 
               
               
                   
               
               
                 311 
                 SOX17 
                 P 
                 fluor- 
                 300 
               
               
                   
                   
                   
                 GAT GGT(dP)GGGTTGGGTT(T*)TTGTTTTT 
                   
               
               
                   
                   
                   
                 GG-quencher/blocker 
                   
               
               
                   
               
               
                 312 
                 SOX17 
                 P 
                 fluor- 
                 301 
               
               
                   
                   
                   
                 CCAAAAACAAAAACCCAA(C**)CCGACC 
                   
               
               
                   
                   
                   
                 ATC-quencher/blocker 
                   
               
               
                   
               
               
                 313 
                 CDO1 
                 P 
                 fluor- 
                 302 
               
               
                   
                   
                   
                 (C*)GTATATTTT(dP)GGTT(T*)TTT(N*)GG 
                   
               
               
                   
                   
                   
                 GT(T*)TC G-quencher/blocker 
                   
               
               
                   
               
               
                 314 
                 CDO1 
                 P 
                 fluor- 
                 303 
               
               
                   
                   
                   
                 C(dK)AAACC(C**)GAAAAAA(C**)C(N*)A 
                   
               
               
                   
                   
                   
                 AAATATAC-quencher/blocker 
                   
               
               
                   
               
               
                 315 
                 TAC1 
                 P 
                 fluor- 
                 304 
               
               
                   
                   
                   
                 GGTAGTTGT(dP)G(T*)CGGGAAGGAGGTT 
                   
               
               
                   
                   
                   
                 CG-quencher/blocker 
                   
               
               
                   
               
               
                 316 
                 TAC1 
                 P 
                 fluor- 
                 305 
               
               
                   
                   
                   
                 C(dK)AACCTCCTTCCCGAC(N*)ACAAC(T*) 
                   
               
               
                   
                   
                   
                 ACC-quencher/blocker 
                   
               
               
                   
               
               
                 317 
                 SOX17 
                 P 
                 fluor- 
                 306 
               
               
                   
                   
                   
                 GGTTTTTTTTGTA(T*)AGATGTGGT(T*)A 
                   
               
               
                   
                   
                   
                 ATGG-quencher/blocker 
                   
               
               
                   
               
               
                 318 
                 SOX17 
                 P 
                 fluor- 
                 307 
               
               
                   
                   
                   
                 CCATTAACCACA(T*)CTA(T*)ACAAAAAA 
                   
               
               
                   
                   
                   
                 AACC-quencher/blocker 
                   
               
               
                   
               
               
                 319 
                 SOX17 
                 E 
                 GGTTTGGTTTATAG(dP)GTATTTAGG 
                 308 
               
               
                   
               
               
                 320 
                 ACTB 
                 I 
                 GAGGTTTAG(T*)AAGTTTTTTGGATTGTG 
                 309 
               
               
                   
               
               
                 321 
                 ACTB 
                 I 
                 CCCTTAAAAAT(T*)ACAAAAACCACAAC 
                 310 
               
               
                   
               
               
                 322 
                 BRCA1 
                 E 
                 GTAGATTGGGTGGTTAATTTAGAG 
                 311 
               
               
                   
               
               
                 323 
                 BRCA1 
                 E 
                 CTATAATTCCC(dK)C(dK)CTTTTC 
                 312 
               
               
                   
               
               
                 324 
                 BRCA1 
                 I 
                 GGTGGTTAATTTAGAGTTTCGAGAGAC 
                 313 
               
               
                   
               
               
                 325 
                 BRCA1 
                 I 
                 CGTTACCACGAAAACCAAAAAACTACCG 
                 314 
               
               
                   
               
               
                 326 
                 BRCA1 
                 P 
                 fluor- 
                 315 
               
               
                   
                   
                   
                 GATTTCGTATTT(T*)GAGAGG(T*)TGTTG 
                   
               
               
                   
                   
                   
                 TTTAG-quencher/blocker 
                   
               
               
                   
               
               
                 327 
                 BRCA1 
                 P 
                 fluor- 
                 316 
               
               
                   
                   
                   
                 CTAAACAACAACC(T*)CTCAAAA(T*)AC 
                   
               
               
                   
                   
                   
                 GAAATC-quencher/blocker 
                   
               
               
                   
               
               
                 328 
                 BRCA1 
                 E 
                 GGTAGATTGGGTGGTTAATTTAGAG 
                 317 
               
               
                   
               
               
                 329 
                 BRCA1 
                 E 
                 CCAAAAAATCTCAACRAACTC 
                 318 
               
               
                   
               
               
                 330 
                 BRCA1 
                 I 
                 GGGTGGTTAATTTAGAGTTTCGAGAGAC 
                 319 
               
               
                   
               
               
                 331 
                 BRCA1 
                 I 
                 ACCACGAAAACCAAAAAACTACCG 
                 320 
               
               
                   
               
               
                 336 
                 MGMT 
                 E 
                 GGGATTTTGTTTAAGTATGTTAAAGG 
                 321 
               
               
                   
               
               
                 337 
                 MGMT 
                 E 
                 CCTACCTTACCTCTAAATACCAACC 
                 322 
               
               
                   
               
               
                 338 
                 MGMT 
                 I 
                 GTATGTTAAAGGGTTGTTGTAAGTTAAG 
                 323 
               
               
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 339 
                 MGMT 
                 I 
                 CCTCTAAATACCAACCCCAAACC 
                 324 
               
               
                   
               
               
                 340 
                 MGMT 
                 P 
                 fluor-CCAACTACTC(C- 
                 325 
               
               
                   
                   
                   
                 quencher)AAAAAACTTCCAAAAACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 341 
                 MGMT 
                 P 
                 fluor-CCAAC(T*)ACTC(C- 
                 326 
               
               
                   
                   
                   
                 quencher)AAAAAAC(T*)TCCAAAAACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 342 
                 MGMT 
                 I 
                 GTATGTTAAAGGGTTGT(T*)GTAAGTTAA 
                 327 
               
               
                   
                   
                   
                 GG 
                   
               
               
                   
               
               
                 343 
                 MGMT 
                 I 
                 CCTCTAAATACCAA(C**)CCCAAACC 
                 328 
               
               
                   
               
               
                 380 
                 ACTB 
                 P 
                 fluor- 
                 329 
               
               
                   
                   
                   
                 CCACCACCCAACACACAA(T*)AACAAAC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 381 
                 ACTB 
                 P 
                 fluor- 
                 330 
               
               
                   
                   
                   
                 CCACCACCCAACACACAA(T*)AACAAAC 
                   
               
               
                   
                   
                   
                 AC-quencher/blocker 
                   
               
               
                   
               
               
                 382 
                 SOX17 
                 I 
                 ATTTAGAGCGCGTTGTTCGC 
                 331 
               
               
                   
               
               
                 383 
                 SOX17 
                 I 
                 ATATCCCCGATAAAACTCAACGACTCG 
                 332 
               
               
                   
               
               
                 384 
                 SOX17 
                 I 
                 TATCCCCGATAAAACTCAACGACTCG 
                 333 
               
               
                   
               
               
                 385 
                 SOX17 
                 I 
                 ATCCCCGATAAAACTCAACGACTCG 
                 334 
               
               
                   
               
               
                 386 
                 TAC1 
                 I 
                 GCGTTCGGATTTTTTTTTCGGC 
                 335 
               
               
                   
               
               
                 387 
                 TAC1 
                 I 
                 TTTCTCTAATTCCTCCGAACGCACG 
                 336 
               
               
                   
               
               
                 388 
                 TAC1 
                 I 
                 CTCTAATTCCTCCGAACGCACG 
                 337 
               
               
                   
               
               
                 389 
                 SOX17 
                 I 
                 GTGACGATTAGAGTTAGATTTAGAGCGC 
                 338 
               
               
                   
               
               
                 390 
                 TAC1 
                 P 
                 fluor- 
                 339 
               
               
                   
                   
                   
                 GTAGTTATCGAGAG(T*)GCGGAGCGA(T*) 
                   
               
               
                   
                   
                   
                 TAG-quencher/blocker 
                   
               
               
                   
               
               
                 391 
                 SOX17 
                 P 
                 fluor- 
                 340 
               
               
                   
                   
                   
                 CCAACCCGACCATCACCGCGAACAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 392 
                 BG converted 
                 I 
                 GGAGAGTGTATTTG(T*)GAAGGAAATG 
                 341 
               
               
                   
               
               
                 393 
                 BG converted 
                 I 
                 CATACATACACCAAACAA(T*)TCATTC 
                 342 
               
               
                   
               
               
                 394 
                 BG converted 
                 P 
                 fluor- 
                 343 
               
               
                   
                   
                   
                 CCAAATACCA(T*)AACCATTTTATTAA(T*) 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 395 
                 BG converted 
                 P 
                 fluor- 
                 344 
               
               
                   
                   
                   
                 CCAAATACCA(T*)AACCATTTTATTAA(T*) 
                   
               
               
                   
                   
                   
                 AACAC-quencher/blocker 
                   
               
               
                   
               
               
                 396 
                 GSTP1 (Fwd) 
                 E 
                 GGTTTYGTTGGGGATTTG 
                 345 
               
               
                   
               
               
                 397 
                 GSTP1 (Fwd) 
                 E 
                 ACCRCTCTTCTAAAAAATCC 
                 346 
               
               
                   
               
               
                 398 
                 GSTP1 (Fwd) 
                 I 
                 AGGTTTTTTCGGTTAGTTGCGC 
                 347 
               
               
                   
               
               
                 399 
                 GSTP1 (Fwd) 
                 I 
                 AACGTCGACCGCAAAAAAACG 
                 348 
               
               
                   
               
               
                 400 
                 GSTP1 (Fwd) 
                 P 
                 fluor-(C*)GCGAT(T*)T(C- 
                 349 
               
               
                   
                   
                   
                 quencher)GGGGA(T*)T(T*)TAGG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 401 
                 GSTP1 (Fwd) 
                 P 
                 fluor-CC(T*)AAAA(T*)C(C- 
                 350 
               
               
                   
                   
                   
                 quencher)CCGAAA(T*)CGC-quencher/blocker 
                   
               
               
                   
               
               
                 402 
                 APC (Fwd) 
                 E 
                 GAAGTAGTTGTGTAATTYGTTGG 
                 351 
               
               
                   
               
               
                 403 
                 APC (Fwd) 
                 E 
                 CACCCTAACRAACTACACC 
                 352 
               
               
                   
               
               
                 404 
                 APC (Fwd) 
                 I 
                 TGCGGATTAGGGCGTTTTTTATTTTC 
                 353 
               
               
                   
               
               
                 405 
                 APC (Fwd) 
                 I 
                 TACAACCACATATCGATCACGTACG 
                 354 
               
               
                   
               
               
                 406 
                 APC (Fwd) 
                 P 
                 fluor-GGAGTTCGTCGA(T*)TGG(T*)TGGG- 
                 355 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 407 
                 APC (Fwd) 
                 P 
                 fluor-CCCAACCAA(T*)CGACGAAC(T*)CC- 
                 356 
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 408 
                 EYA4 (Fwd) 
                 E 
                 GAGTTTTTYGGAGGGTTATAG 
                 357 
               
               
                   
               
               
                 409 
                 EYA4 (Fwd) 
                 E 
                 CAAACTACAAAAAACATTCAATCC 
                 358 
               
               
                   
               
               
                 410 
                 EYA4 (Fwd) 
                 I 
                 GCGTTTGGGTTTTTTCGGTGTC 
                 359 
               
               
                   
               
               
                 411 
                 EYA4 (Fwd) 
                 I 
                 ATCGCCGCAATTAAAAAACCCG 
                 360 
               
               
                   
               
               
                 412 
                 EYA4 (Fwd) 
                 P 
                 fluor- 
                 361 
               
               
                   
                   
                   
                 GGTTCGCGTTTTAAT(T*)TTTAGG(T*)ATT 
                   
               
               
                   
                   
                   
                 G-quencher/blocker 
                   
               
               
                   
               
               
                 413 
                 EYA4 (Fwd) 
                 P 
                 fluor- 
                 362 
               
               
                   
                   
                   
                 CAATACC(T*)AAAAAT(T*)AAAACGCGA 
                   
               
               
                   
                   
                   
                 ACC-quencher/blocker 
                   
               
               
                   
               
               
                 414 
                 OLIG2(Fwd) 
                 E 
                 GTTATGGATTYGGAYGTTAG 
                 363 
               
               
                   
               
               
                 415 
                 OLIG2(Fwd) 
                 E 
                 CTCCRACRAACAATCACTC 
                 364 
               
               
                   
               
               
                 416 
                 OLIG2(Fwd) 
                 I 
                 GTTTGGTGTTTAG(T*)CGTTCGTC 
                 365 
               
               
                   
               
               
                 417 
                 OLIG2(Fwd) 
                 I 
                 CACTCGAAATAAA(C**)GAAAACACG 
                 366 
               
               
                   
               
               
                 418 
                 OLIG2(Fwd) 
                 P 
                 fluor- 
                 367 
               
               
                   
                   
                   
                 GGTAGTAGCGG(T*)AGCGTT(T*)TTATTG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 419 
                 OLIG2(Fwd) 
                 P 
                 fluor- 
                 368 
               
               
                   
                   
                   
                 CAATAAAAACGC(T*)ACCGC(T*)ACTACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 420 
                 ADAMTS12(Fwd) 
                 E 
                 GGYGTAGTTTATTTYGGTT 
                 369 
               
               
                   
               
               
                 421 
                 ADAMTS12(Fwd) 
                 E 
                 ATTTAACCRACTCRACCAAC 
                 370 
               
               
                   
               
               
                 422 
                 ADAMTS12(Fwd) 
                 I 
                 GTATGTTTCGCGGTTTCGTAGTTC 
                 371 
               
               
                   
               
               
                 423 
                 ADAMTS12(Fwd) 
                 I 
                 ACTAAACCTAACG(T*)TCGAAACG 
                 372 
               
               
                   
               
               
                 424 
                 ADAMTS12(Fwd) 
                 P 
                 fluor-(C*)GTTCGTTCGG(T- 
                 373 
               
               
                   
                   
                   
                 quencher)G(T*)ATTTTTT(T*)TTCGG- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 425 
                 ADAMTS12(Fwd) 
                 P 
                 fluor-CCGAAAAAAAAA(T- 
                 374 
               
               
                   
                   
                   
                 quencher)A(C**)ACCGAA(C**)GAAC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 426 
                 POU4F1(Fwd) 
                 E 
                 GTTTGAGTTGTTTTGATTTTAGTG 
                 375 
               
               
                   
               
               
                 427 
                 POU4F1(Fwd) 
                 E 
                 CTCCAACCTCAACTCTAAAC 
                 376 
               
               
                   
               
               
                 428 
                 POU4F1(Fwd) 
                 I 
                 GATTTTAGTGTCGCGTATTTTGGTTC 
                 377 
               
               
                   
               
               
                 429 
                 POU4F1(Fwd) 
                 I 
                 CTAAACTAAATCCCGCGAACCTCG 
                 378 
               
               
                   
               
               
                 430 
                 POU4F1(Fwd) 
                 P 
                 fluor- 
                 379 
               
               
                   
                   
                   
                 GGTTTTAT(T*)GGGGGTT(N*)AT(T*)TCG 
                   
               
               
                   
                   
                   
                 GGTAG-quencher/blocker 
                   
               
               
                   
               
               
                 431 
                 POU4F1(Fwd) 
                 P 
                 fluor- 
                 380 
               
               
                   
                   
                   
                 CTACCCGAAATAACCC(C**)CAA(N*)TAA 
                   
               
               
                   
                   
                   
                 AA(C**)C-quencher/blocker 
                   
               
               
                   
               
               
                 432 
                 ABCB1(Fwd) 
                 E 
                 GGTTTTTAGTATTTTTAYGAAGGT 
                 381 
               
               
                   
               
               
                 433 
                 ABCB1(Fwd) 
                 E 
                 CRATACRAAAACCTACTCTCTA 
                 382 
               
               
                   
               
               
                 434 
                 ABCB1(Fwd) 
                 I 
                 TTTGGATTTTGTTCGTCGTTAGTGC 
                 383 
               
               
                   
               
               
                 435 
                 ABCB1(Fwd) 
                 I 
                 CTACTCTCTAAACCCGCGAACG 
                 384 
               
               
                   
               
               
                 436 
                 ABCB1(Fwd) 
                 P 
                 fluor- 
                 385 
               
               
                   
                   
                   
                 GGTTTTAGTCG(T*)CGCGGACGATGT- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 437 
                 ABCB1(Fwd) 
                 P 
                 fluor- 
                 386 
               
               
                   
                   
                   
                 ACATCGTCCGCGACGAC(T*)AAAACC- 
                   
               
               
                   
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 438 
                 SOX17 
                 I 
                 GAGTTAGATTTAGAGCGCGTTGTTC 
                 387 
               
               
                   
               
               
                 439 
                 TAC1 
                 I 
                 GAGCGCGTTCGGATTTTTTTTTC 
                 388 
               
               
                   
               
            
           
         
       
     
     It is noted that these primers and probes identify the locations of various fluorophores and quenchers. However, it will be recognized that the particular fluorophores and quenchers are illustrative and not limiting and numerous amplification and/or detection strategies can be employed in the cartridges described herein. Accordingly, in various embodiments the methods and devices described herein can employ many different nucleic acid hybridization probes. Typically, for signal generation, the probes utilize a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety brought about by changing the distance between the fluorophore and the interacting molecule or moiety. Alternatively, other methods of detecting a polynucleotide in a sample, including, but not limited to, the use of radioactively-labeled probes, are contemplated. 
     Fluorescence-based assays typically rely for signal generation on fluorescence resonance energy transfer, or “FRET”, according to which a change in fluorescence is caused by a change in the distance separating a first fluorophore from an interacting resonance energy acceptor, either another fluorophore or a quencher. Combinations of a fluorophore and an interacting molecule or moiety, including quenching molecules or moieties, are known as “FRET pairs.” The mechanism of FRET-pair interaction typically requires that the absorption spectrum of one member of the pair overlaps the emission spectrum of the other member, the first fluorophore. If the interacting molecule or moiety is a quencher, its absorption spectrum typically overlaps the emission spectrum of the fluorophore (see, e.g., Stryer (1978)  Ann. Rev. Biochem.  47: 819-846; Selvin (1995)  Meth. Enzymol.  246: 300-335; and the like). Efficient FRET interaction is typically achieved when the absorption and emission spectra of the pair have a large degree of overlap. The efficiency of FRET interaction is linearly proportional to that overlap. Typically, a large magnitude of signal (i.e., a high degree of overlap) is desired. FRET pairs, including fluorophore-quencher pairs, are therefore typically chosen on that basis. 
     A variety of labeled nucleic acid hybridization probes and detection assays that utilize FRET and FRET pairs are known. One such scheme is described by Cardullo et al. (1988)  Proc. Natl. Acad. Sci. USA,  85: 8790-8794and in Heller et al. EP 0070685. It uses a probe comprising a pair of oligodeoxynucleotides complementary to contiguous regions of a target DNA strand. One probe molecule contains a fluorescent label, a fluorophore, on its 5′ end, and the other probe molecule contains a different fluorescent label, also a fluorophore, on its 3′ end. When the probe is hybridized to the target sequence, the two labels are brought very close to each other. When the sample is stimulated by light of an appropriate frequency, fluorescence resonance energy transfer from one label to the other occurs. FRET produces a measurable change in spectral response from the labels, signaling the presence of targets. One label could be a “quencher,” which can be, inter alia, an interactive moiety (or molecule) that releases the accepted energy as heat. 
     Another type of nucleic acid hybridization probe assay utilizing a FRET pair is the “TaqMan®” assay described in Gelfand et al. U.S. Pat. No. 5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. The probe is typically a single-stranded oligonucleotide labeled with a FRET pair. In a TaqMan® assay, a DNA polymerase releases single or multiple nucleotides by cleavage of the oligonucleotide probe when it is hybridized to a target strand. That release provides a way to separate the quencher label and the fluorophore label of the FRET pair. 
     In certain embodiments non-FRET fluorescent probes, such as those described in, e.g., Tyagi et al., U.S. Pat. No. 6,150,097 can also be used. For example, the Tiyagi et al. patent describes how changes in the absorption spectra of the label pair can be used as a detectable signal as an alternative to change in fluorescence. When change in absorption is utilized, the label pair may include any two chromophores, that is, fluorophores, quenchers and other chromophores. The label pair may even be identical chromophores. 
     In some embodiments, dyes and other moieties, such as quenchers, are introduced into primers and/or probes used in the methods and cartridges described herein. In certain embodiments such dyes and quenchers include, but are not limited to dyes (fluors) suitable for use as FRET probes. In certain embodiments the dyes and/or quenchers comprise modified nucleotides. A “modified nucleotide” refers to a nucleotide that has been chemically modified, but still functions as a nucleotide. In some embodiments, the modified nucleotide has a chemical moiety, such as a dye or quencher, covalently attached, and can be introduced into a polynucleotide, for example, by way of solid phase synthesis of the polynucleotide. In some embodiments, the modified nucleotide includes one or more reactive groups that can react with a dye or quencher before, during, or after incorporation of the modified nucleotide into the nucleic acid. In some embodiments, the modified nucleotide is an amine-modified nucleotide, i.e., a nucleotide that has been modified to have a reactive amine group. In some embodiments, the modified nucleotide comprises a modified base moiety, such as uridine, adenosine, guanosine, and/or cytosine. In some embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many amine modified nucleotides are commercially available from, e.g., Applied Biosystems, Sigma, Jena Bioscience and TriLink. An illustrative, but non-limiting list of suitable fluors is shown in Table 6. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Illustrative, but non-limiting fluorophores 
               
               
                 (fluorescent labels) for use in the 
               
               
                 primers and/or probes described herein. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Absorbance 
                 Emission 
               
               
                   
                 Dye 
                 Wavelength 
                 Wavelength 
               
               
                   
                   
               
               
                   
                 Alexa fluor 
                 345 
                 442 
               
               
                   
                 Alexa fluor 430 
                 430 
                 545 
               
               
                   
                 Alexa fluor 488 
                 494 
                 517 
               
               
                   
                 Alexa fluor 532 
                 530 
                 555 
               
               
                   
                 Alexa fluor 546 
                 556 
                 573 
               
               
                   
                 Alexa fluor 555 
                 556 
                 573 
               
               
                   
                 Alexa fluor 568 
                 578 
                 603 
               
               
                   
                 Alexa fluor 594 
                 590 
                 617 
               
               
                   
                 Alexa fluor 633 
                 621 
                 639 
               
               
                   
                 Alexa fluor 633 
                 650 
                 668 
               
               
                   
                 Alexa fluor 660 
                 663 
                 690 
               
               
                   
                 Alexa fluor 680 
                 679 
                 702 
               
               
                   
                 Allophycocyanin 
                 650 
                 660 
               
               
                   
                 Aminocoumarin 
                 350 
                 445 
               
               
                   
                 Cy2 
                 490 
                 510 
               
               
                   
                 Cy3 
                 550 
                 570 
               
               
                   
                 Cy3.5 581 
                 581 
                 596 
               
               
                   
                 Cy5 
                 650 
                 670 
               
               
                   
                 Cy5.5 
                 675 
                 694 
               
               
                   
                 Cy7 
                 743 
                 770 
               
               
                   
                 FAM 
                 495 
                 516 
               
               
                   
                 Fluorescein FITC 
                 495 
                 518 
               
               
                   
                 HEX 
                 535 
                 556 
               
               
                   
                 Hydroxycoumarin 
                 325 
                 386 
               
               
                   
                 Methoxycoumarin 
                 360 
                 410 
               
               
                   
                 Red 613 
                 480; 565 
                 613 
               
               
                   
                 Rhodamine Red-X 
                 560 
                 580 
               
               
                   
                 Rox 
                 575 
                 602 
               
               
                   
                 R-phycoerythrin (PE) 
                 480; 565 
                 578 
               
               
                   
                 Tamara 
                 565 
                 580 
               
               
                   
                 Texas Red 
                 615 
                 615 
               
               
                   
                 TRITC 
                 547 
                 572 
               
               
                   
                 TruRed 
                 490; 675 
                 695 
               
               
                   
                   
               
            
           
         
       
     
     If the assay is designed to detect one target DNA sequence then only one fluorescent hybridization probe needs to be used and, in certain embodiments, FAM, TET, or HEX (or one of their alternatives listed in Table 7) will be a good fluorophore to label the probe. These fluorophores can readily be excited and detected in various spectrofluorometric thermal cyclers. In addition, because of the availability of phosphoramidites derivatives of these fluorophores and the availability of quencher-linked control-pore glass columns, fluorescent hybridization probes with these labels can be entirely synthesized in an automated DNA synthesis process, with the advantage of relatively less expensive and less labor intensive probe manufacture. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Additional illustrative fluorophore labels 
               
               
                 for fluorescent hybridization probes. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Excitation 
                 Emission 
               
               
                 Fluorophore 
                 Alternative Fluorophore 
                 (nm) 
                 (nm) 
               
               
                   
               
               
                 Cy3 3   
                 NED 2 , Quasar 570 1 , Oyster 556 4   
                 550 
                 570 
               
               
                 Cy5 3   
                 LC red 670 5 , Quasar 670 1 , Oyster 645 4   
                 650 
                 670 
               
               
                 HEX 
                 JOE, VIC  B , CAL Fluor Orange 560 1   
                 535 
                 555 
               
               
                 LC red 640 5   
                 CAL Fluor Red 635  A   
                 625 
                 640 
               
               
                 LC red 705 5   
                 Cy5.5 3   
                 680 
                 710 
               
               
                 ROX 
                 LC red 610 5 , CAL Fluor Red 610 1   
                 575 
                 605 
               
               
                 TET 
                 CAL Fluor Gold 540 1   
                 525 
                 540 
               
               
                 Texas red 
                 LC red 610 5 , CAL Fluor Red 610 1   
                 585 
                 605 
               
               
                 TMR 
                 CAL Fluor Red 590 1   
                 555 
                 575 
               
               
                   
               
               
                   1 CAL and Quasar fluorophores are available from Biosearch Technologies; 
               
               
                   2 VIC and NED are available from Applied Biosystems; 
               
               
                   3 Cy dyes are available from Amersham Biosciences; 
               
               
                   4 Oyster fluorophores are available from Integrated DNA Technologies; and 
               
               
                   5 LC (Light Cycler) fluorophores are available from Roche Applied Science. 
               
            
           
         
       
     
     In certain embodiments, multiple target genes are detected in a single multiplex reaction. In some embodiments, each probe that is targeted to a different gene is spectrally distinguishable (detectably different) from the other probes utilized in the multiplex reaction. Probe combinations suitable for multiplex detection are known to those of skill in the art. For example, illustrative combinations of detectably different fluorphores in four target multiplex systems include, but are not limited to: 
     1) FAM, TMR, Texas red, and Cy5; 
     2) FAM, TET, TMR, and Texas Red; 
     3) FAM, HEX, Texas red, and Cy5; and 
     4) FAM, Cy3, Texas red, and Cy5. 
     An illustrative combination of detectably different fluorphores in a five target multiplex systems is FAM, TET, TMR, Texas Red, and Cy5. Illustrative combinations of detectable different fluorophores in a six target multiplex system include, but are not limited to: 
     1) FAM, TET, HEX, TMR, ROX, and Texas red; and 
     2) FAM, HEX, LC red 610, LC red 640, LC red 670, and LC red 705. 
     It will be recognized that these combinations of fluorophores are illustrative and non-limiting and numerous other fluorophores will be available to those of skill in the art. 
     As noted above, for the design of fluorescent hybridization probes that utilize fluorescence resonance energy transfer (FRET), fluorophore-quencher pairs that have sufficient spectral overlap should be chosen. Fluorophores with an emission maximum between 500 and 550 nm, such as FAM, TET and HEX, are best quenched by quenchers with absorption maxima between 450 and 550 nm, such as dabcyl, BHQ-1, and the like (see, e.g., Table 8 for illustrative quencher labels). Fluorophores with an emission maximum above 550 nm, such as rhodamines (including TMR, ROX and Texas red) and Cy dyes (including Cy3 and Cy5) are effectively quenched by quenchers with absorption maxima above 550 nm (including BHQ-2). 
     For the design of fluorescent hybridization probes that utilize contact quenching, any non-fluorescent quencher can serve as a good acceptor of energy from the fluorophore. For example, Cy3 and Cy5 are effectively quenched by the BHQ-1 and BHQ-2 quenchers. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Illustrative quencher labels for fluorescent hybridization probes. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Absorption Maximum 
               
               
                   
                 Quencher 
                 (nm) 
               
               
                   
                   
               
               
                   
                 BHQ-1 4   
                 534 
               
               
                   
                 BHQ-2 4   
                 580 
               
               
                   
                 BHQ-3 4   
                 670 
               
               
                   
                 Dabcyl 
                 475 
               
               
                   
                 DDQ-I 1   
                 430 
               
               
                   
                 DDQ-II 1   
                 630 
               
               
                   
                 Eclipse 2   
                 530 
               
               
                   
                 Iowa Black FQ 3   
                 532 
               
               
                   
                 Iowa Black RQ 3   
                 645 
               
               
                   
                 QSY-21 5   
                 660 
               
               
                   
                 QSY-7 5   
                 571 
               
               
                   
                   
               
               
                   
                   1 DDQ or Deep Dark Quenchers are available from Eurogentec; 
               
               
                   
                   2 Eclipse quenchers are available from Epoch Biosciences; 
               
               
                   
                   3 Iowa quenchers are available from Integrated DNA Technologies; 
               
               
                   
                   4 BHQ or Black Hole quenchers are available from Biosearch Technologies; and 
               
               
                   
                   5 QSY quenchers are available from Molecular Probes. 
               
            
           
         
       
     
     In certain embodiments nucleotides can quench the fluorescence of fluorophores, with guanosine being the most efficient quencher, followed by adenosine, cytidine and thymidine. In general, fluorophores with an excitation wavelength between 500 and 550 nm are quenched more efficiently by nucleotides than fluorophores with longer excitation wavelengths. In designing fluorescent hybridization probes, it can be desirable to avoid placing a fluorophore label directly next to a guanosine, to ensure higher fluorescence signals from the fluorophore. 
     The stabilizing effect of some fluorophore-quencher pairs that interact by contact quenching can have important consequences for the design of hybridization probes (see, e.g., Marras et al. (2002)  Nucleic Acids Res.  30: e122; Johansson et al. (2002)  J. Am. Chem. Soc.  124: 6950-6956). For example, it has been observed that hybridization probes labeled with a fluorophore quenched by either BHQ-1 or BHQ-2 show an increase in hybrid melting temperature of about 4° C., compared to hybridization probes with the same probe sequence, but labeled with fluorophores quenched by dabcyl. It is also noted that strong affinity has been observed between the Cy dyes, Cy3 and Cy5, and the Black Hole quenchers, BHQ-1 and BHQ-2. 
     In view of the foregoing and the Examples and teachings provided herein, numerous primer/probe combinations will be available for use in the methods and cartridges described herein. 
     Cartridge, Modules, and Systems for DNA Methylation Analysis. 
     In certain embodiments cartridges are provided for performing the methods described herein (e.g., determination of DNA methylation and, optionally RNA expression). In certain embodiments the cartridge comprises a column comprising a first matrix material, a sample receiving chamber, a temperature controlled channel or chamber, a plurality of chambers containing reagents and/or buffers, and when in use at least one of said chambers contains a DNA conversion reagent (e.g., DABSO and/or a bisulfite reagent), and at least one of said chambers contains a desulphonation/elution buffer, and wherein said cartridge optionally comprises a second column comprising said second matrix material. In certain embodiments the cartridge is configured so that in use, the cartridge comprises a chamber containing a reagent comprising guanidinium thiocyanate ethanol (GTC-EtOH). In certain embodiments the second column is absent, while in other embodiments the second column is present. In certain embodiments the temperature controlled channel or chamber can simply be a heating channel or chamber, or it can be a thermocycling channel or chamber. In certain embodiments the cartridge further comprises a second heating channel or chamber (e.g., a second thermocycling channel or chamber). In certain embodiments the cartridge is configured so that a DNA conversion step (e.g., bisulfite incubation) and/or a desulphonation step occurs in the same reaction tube or chamber in which one or more PCR reactions are later performed. 
     In certain embodiments the bisulfite reagent is provided as a component of the cartridge. In certain other embodiments the cartridge is configured for the bisulfite reagent to be added to the cartridge at or near the time the sample is placed in the cartridge. In certain instances, the bisulfite reagent is added directly into a chamber in the cartridge, while in other embodiments, the bisulfite reagent is introduced into a loading port on the cartridge (e.g., an injection port) to introduce the bisulfite reagent into the cartridge. In certain embodiments the bisulfite reagent is introduced into the cartridge by the system operating the cartridge (e.g., a processing module) while the cartridge is operating to determine DNA methylation. 
     In certain embodiments the reagent comprising guanidinium thiocyanate (e.g., GTC-EtOH) is provided as a component of the cartridge. In certain other embodiments the cartridge is configured for the reagent comprising guanidinium thiocyanate to be added to the cartridge at or near the time the sample is placed in the cartridge. In certain instances, the reagent comprising guanidinium thiocyanate is added directly into a chamber in the cartridge, while in other embodiments, the reagent comprising guanidinium thiocyanate is introduced into a loading port on the cartridge (e.g., an injection port) to introduce the bisulfite reagent into the cartridge. In certain embodiments the reagent comprising guanidinium thiocyanate is introduced into the cartridge by the system operating the cartridge (e.g., a processing module) while the cartridge is operating to determine DNA methylation. 
     In various illustrative, but non-limiting embodiments, the conversion reagent (e.g., bisulfite reagent) comprises a compound selected from the group consisting of sodium metabisulfite, potassium bisulfite, cesium bisulfite, DABSO, and ammonium bisulfite. In certain embodiments the bisulfite is provided in a reagent mix comprising scavengers (e.g., TROLOX water-soluble analog of vitamin E, hydroquinone, etc.) to prevent sulfite oxidation and/or catalysts. In certain embodiments the bisulfite is provided in a reagent mix comprising polyamines as catalysts. 
     In various embodiments the first matrix material and/or said second matrix material, when present, comprises a material selected from the group consisting of glass or silica, an ion exchange resin, and hydroxyapatite. 
     In various embodiments the cartridge comprises one or more chambers (e.g., 1 chamber, 2 chambers, 3 chambers, 4 chambers, etc.) each containing one or more reagents selected from the group consisting of methylation specific PCR primers, methylation specific PCR probes, PCR enzyme(s) (e.g., polymerase), reverse transcriptase, and PCR reaction buffer. 
     In certain embodiments the cartridge contains one or more chambers containing primers specific for bisulfite-converted methylated and/or unmethylated sequences. In certain embodiments the cartridge comprises one or more chambers containing primers and probes for a MethyLight PCR protocol. In certain embodiments the cartridge comprises one or more chambers containing reagents for TaqMan PCR reactions. In certain embodiments the cartridge comprises one or more chambers containing one or more fluorescent probes that are markers for amplified methylated sequences and/or one or more fluorescent probes that are markers for amplified unmethylated sequences. In certain embodiments the probes comprise a fluorescent reporter dye and a quencher dye, where the probes provides a signal upon cleavage by the 5′ to 3′ nuclease activity of Taq DNA polymerase. In certain embodiments the cartridge comprises a plurality of probes each specific to a different methylated region in an amplified region of interest. In certain embodiments the cartridge comprises a single probe specific to a methylated region in an amplified region of interest. In certain embodiments the cartridge comprises a plurality of probes each specific to the same methylated region in an amplified region of interest. 
     Illustrative primers and probes include, but are not limited to primers and/or probes to determine methylation of a promoter region of a gene selected from the group consisting of APC, ARF, CDKN2B, CDKN2A, BRCA1, VLH, hMLH1, MGMT. RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, AKR1B1, HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. In certain embodiments the primers and/or probes are selected to determine methylation of a promoter region of a gene selected from the group consisting of MGMT, RASSF1A, ADAMTS1, BNC1, HIST1H3C, HOXB4, RASGRF2, TM6SF1, and AKR1B1. In various embodiments the PCR primers, and/or probes, and/or enzymes are provided as beads, e.g., as described in U.S Patent Publication No: 2006/0068399, which is incorporated herein by reference for the beads and bead formulations described therein. 
     In various embodiments the cartridge is configured so that the sample receiving chamber, said column(s), the plurality of chambers, and the temperature controlled channel or chamber, are selectively in fluid communication. In certain embodiments the selective fluid communication is provided by microfluidic channels and valves. In certain embodiments the selective fluid communication is provided by providing the sample receiving chamber, said column(s), said plurality of chambers, the heating channel or chamber or a port into the heating channel or chamber, disposed around a central valve and selectively in fluid communication with a channel in said central valve. 
     In certain embodiments the cartridge is configured so that, when in use, the cartridge comprises: a first chamber containing a sample; a second chamber containing a guanidinium thiosulfate-ethanol (GTC-EtOH) solution; a third chamber containing a bisulfate reagent; a fourth chamber containing a buffer; a fifth chamber containing a rinse solution; and a sixth chamber containing an elution/desulphonation reagent. In certain embodiments the cartridge comprises a seventh chamber containing PCR primers and/or probes and/or PCR enzymes. In certain embodiments the cartridge comprises an eighth chamber also containing PCR primers and/or probes and/or PCR enzymes. 
       FIGS. 1A, 1B and 2  illustrate one cartridge suitable for practice of the methods described herein. The illustrated cartridges are based on the GENEXPERT® cartridge (Cepheid, Inc., Sunnyvale, Calif.). As shown in  FIG. 2 , panel A the cartridge  200  comprises a cartridge body  202  containing a plurality of reagent and/or buffer chambers  208 . The chambers are disposed around a central syringe barrel  206  that is in fluid communication with a valve body  210  (panel B and  FIG. 1B ) and that is sealed with a gasket  204 . The valve body  210  can comprise a cap  212  and the entire cartridge body can be supported on a cartridge base  226 . A “plunger” not shown can be operated to draw fluid into the syringe barrel  206  and rotation of the syringe barrel  206  and associated valve body  212  provides selective fluid communication between the chambers  208  a cavity  214  that can contain a matrix material as described herein and function as a column. In various embodiments the cartridge further comprises one or more temperature controlled channels or chambers  216  that can, in certain embodiments, function as thermocycling chambers. The temperature controlled channels or chambers are also selectively in fluid communication with the cavity  214  and/or the chambers  208 . As shown in  FIG. 1A , in certain embodiments, the cartridge provides optical windows to provide real-time detection of, e.g., amplification products, base identity in sequencing operations, and the like. 
     In certain embodiments the cartridge  200  is configured for insertion into a reaction module  300 , e.g., as shown in  FIG. 3A . As illustrated in  FIG. 3B  the module is configured to receive the cartridge  200 . In certain embodiments the reaction module provides heating plates  308  to heat the temperature controlled chamber or channel. The module can optionally additionally include a fan  304  to provide cooling where the temperature controlled channel or chamber is a thermocycling channel or chamber. Electronic circuitry  302  can be provided to pass information (e.g., optical information) top a computer for analysis. In certain embodiments the module can contain optical blocks  306  to provide excitation and/or detection of one or more (e.g., 1, 2, 3, 4, or more) optical signals representing, e.g., various nucleic acid targets. In various embodiments an electrical connector  312  can be provided for interfacing the module with a system (e.g. system controller or with a discrete analysis/controller unit. As illustrated, in  FIG. 3B  the sample can be introduced into the cartridge using a pipette  310 . 
     In certain embodiments, the module also contains a controller that operates a plunger in the syringe barrel and the rotation of the valve body. 
     In certain embodiments a system (e.g., a processing unit) is provided. One illustrative, but non-limiting embodiment is shown in  FIG. 3C . In certain embodiments, the processing unit comprises an enclosure configured to contain one or more sample processing modules where each processing module is configured to hold and operate a removable cartridge as described herein. In certain embodiments the system is configured to operate the sample processing modules to perform sample processing to determine methylation of one or more target nucleic acids and optionally to determine the level of one or more target RNA/DNA sequences within a corresponding removable sample cartridge, wherein the processing on a sample within the corresponding removable sample cartridge performs a method as described herein. In certain embodiments the system is configured to contain one sample processing module. In certain embodiments the system is configured to contain at least two sample processing modules, or at least 4 sample processing modules, or at least 8 sample processing modules, or at least 12 sample processing modules, or at least 16 sample processing modules, or at least 20 sample processing modules, or at least 24 sample processing modules, or at least 28 sample processing modules, or at least 32 sample processing modules, or at least 64 sample processing modules, or at least 128 sample processing modules. In certain embodiments the system provides a user interface that allows the user input operational instructions and/or to monitor operation of the cartridges to determine DNA methylation. 
     While the methods described herein are described primarily with reference to the GENEXPERT® cartridge by Cepheid Inc. (Sunnyvale, Calif.) (see, e.g.,  FIG. 1A ), it will be recognized, that in view of the teachings provided herein the methods can be implemented on other cartridge/microfluidic systems. Such cartridge/microfluidic systems can include, for example microfluidic systems implemented using soft lithography, micro/nano-fabricated microfluidic systems implemented using hard lithography, and the like. 
     High Volume Sample Preparation (HVSP) Cartridge. 
     In various embodiments cartridges are provided for the preparation of large sample volumes. In certain embodiments the sample preparation cartridges comprise GENEXPERT® cartridges modified for high volume sample preparation (e.g., as shown in  FIG. 20 ). In certain embodiments, e.g., when the cartridge is based on a GENEXPERT® cartridge comprises one or more channels or chambers comprising an affinity matrix that binds DNA, a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with said central valve assembly where the central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with the central valve wherein said plurality of chambers comprises at least two different chambers each configured to receive up to about 4 ml (or up to about 5 ml) of sample solution (in certain embodiments chamber 2 has a maximum volume of about 4 ml, while chamber 3 has a maximum volume of about 4.5 ml), a chamber containing PEG (e.g., PEG200), a chamber containing an alkaline solution (e.g., KOH solution), and a chamber containing a buffer (e.g., Tris). In certain embodiments the plurality of chambers comprises at least three different chambers each configured to receive up to about 4 ml (or up to about 5 ml) of sample solution. In certain embodiments the plurality of chambers comprises a chamber containing a wash solution (e.g., GTC-ethanol wash solution which is typically 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, 50% ethanol). In certain embodiments the cartridge comprises a chamber configured for removal of a processed sample. In certain embodiments the sample chambers, when in use, contain sample solution, GTC and alcohol(e.g., isopropanol). In certain embodiments the sample chambers, when in use contain sample solution, GTC and alcohol in substantially equal volumes. In certain embodiments the cartridge, when in use, comprises 4 ml of sample solution GTC and isopropanol disposed in each of said chambers configured to receive a sample. In certain embodiments the cartridge provides DNA or RNA recovery that is substantially linear with respect to the sample volume between 0.5 ml and about 4 ml of sample. 
     In certain embodiments the HVSP cartridge is configured to perform a DNA conversion (e.g., bisulfite conversion) to provide a methylation analysis. Accordingly in certain embodiments, the HVSP cartridge is configured to contain, or to receive immediately or shortly prior to use, a conversion reagent (e.g. a bisulfite reagent, DABSO, etc.). In certain embodiments, the HVSP cartridge can be configured to also contain reagents for and to provide a desulphonation of converted DNA. Alternatively, in certain embodiments, the conversion is performed in the HSVP cartridge while the desulphonation and methylation analysis (e.g., PCR) is performed in the second cartridge (e.g., as illustrated in the work flows shown in  FIG. 20B ). 
     Two-Cartridge Methylation Analysis 
     In various embodiments the methylation analysis methods described herein are performed using a two-cartridge system (a set of two cartridges such as a “first” cartridge (e.g., a high volume sample preparation cartridge), and a “second” cartridge (e.g., a qPCR cartridge)), e.g., as illustrated in  FIG. 20B . As illustrated in this figure, in certain embodiments, sample preparation and bisulfite conversion can be performed in a first cartridge. The converted DNA is then transferred to second cartridge wherein it is washed and desulphonated. In certain embodiments the second cartridge is additionally utilized to analyze the converted (or unconverted) DNA, e.g., via PCR (e.g., methylation-specific PCR). 
     In certain embodiments the first cartridge can be a high-volume sample cartridge as described above where the sample preparation cartridge contains a bisulfite conversion reagent as described herein. In certain embodiments the high-volume cartridge provides a larger sample chamber or multiple sample chambers to permit cleanup and conversion of larger amounts of DNA. 
     In certain embodiments the first cartridge of the two-cartridge set comprises a sample receiving chamber, a “column” comprising a first matrix material, a temperature controlled channel or chamber, a sample removal chamber, and a plurality of chambers containing reagents and/or buffers. Typically, in use, at least one of the chambers contains a bisulfite reagent (e.g., as described herein). In certain embodiments the second cartridge of the two-cartridge set comprises a sample receiving chamber, a “column” comprising a matrix material, a temperature controlled channel or chamber, a plurality of chambers containing reagents and/or buffers. Typically, in use, at least one of the plurality of chambers in the second cartridge contains a desulphonation and/or elution reagent. In certain embodiments the second cartridge contains reagents for PCR amplification (e.g., methylation specific PCR) and the detection of an amplification product. 
     In certain embodiments illustrative, but non-limiting embodiments, the first cartridge is used for sample preparation (e.g., binding of DNA to a first matrix material (e.g., glass fiber), washing and eluting of the bound DNA) and bisulfite conversion of the washed DNA. In certain embodiments the bisulfite conversion comprise combining the washed DNA with a bisulfite reagent and heating the mixture in the temperature-controlled channel or chamber. The resulting converted DNA is then mixed with a wash buffer (e.g., 1.25M GTC, 25 mM Tris pH 7.0, 50% ethanol) and moved into a sample removal chamber (e.g., chamber 2) where it can be removed (e.g., removed using a pipette, a syringe or syringe, pump, and the like). 
     In certain embodiments the bisulfite converted DNA in wash buffer is introduced into a sample receiving chamber of the second cartridge. The second cartridge is operated to bind the DNA to a second matrix material (e.g., glass fiber “column”), to wash and elute the bound DNA and to desulphonate the DNA. In certain embodiments the DNA is desulphonated on the column or as it is eluted from the second matrix material. In certain embodiments the DNA is desulphonated after removal from the second matrix material. In certain embodiments the desulphonated DNA is then mixed, e.g., with enzyme and primer/probe beads for PCR in the second cartridge. In certain embodiments the desulphonated DNA is removed and can be introduced into a third cartridge or other reaction system for further analysis (e.g., sequencing). 
     In certain embodiments, in the first cartridge, the sample receiving chamber, column, plurality of chambers, sample removal chamber, and temperature-controlled heating channel or chamber, are selectively in fluid communication (e.g., by microfluidic channels and/or valves); and/or, in the second cartridge, the sample receiving chamber, the column, the plurality of chambers, and the temperature-controlled heating channel or chamber, are selectively in fluid communication (e.g., by microfluidic channels and/or valves). 
     In certain embodiments, in the first cartridge, the sample receiving chamber, the column, the plurality of chambers, the sample removal chamber, and the temperature controlled channel or chamber or a port into the temperature controlled channel or chamber, are disposed around a central valve and selectively in fluid communication with one or more channels in the central valve. The central valve can be configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber(s) in fluid communication with the central valve. In certain embodiments, in the second cartridge, the sample receiving chamber, the column, the plurality of chambers, and the temperature controlled channel or chamber or a port into the temperature controlled channel or chamber, are disposed around a central valve and selectively in fluid communication with one or more channels in the central valve. In certain embodiments the central valve is configured to accommodate a plunger that is capable of drawing fluid into or out of one or more chambers in fluid communication with the central valve. 
     In certain embodiments the first cartridge and/or the second cartridge is a GENEXPERT® cartridge, or a modified GENEXPERT® cartridge (e.g., as illustrated in  FIGS. 1A, 1B, 2, 13A, 17, and 20A ). In certain, illustrative, but non-limiting embodiments, the first cartridge is configured as shown in Table 9. In certain embodiments the chamber numbering corresponds to the chambers illustrated in  FIG. 1B . 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 One illustrative, but non-limiting configuration of a ″first″ 
               
               
                 cartridge for DNA preparation and bisulfite conversion. 
               
            
           
           
               
               
               
               
            
               
                   
                 Chamber # 
                 Reagent 
                 Volume 
               
               
                   
               
               
                   
                 Chamber 1  
                 Empty 
                 N/A 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Chamber 2  
                 Sample in GTC Tween 
                 2.5  
                 mL 
               
               
                   
                 Chamber 3  
                 1.25M GTC, 25 mM Tris 
                 3.0  
                 mL 
               
               
                   
                   
                 pH 7.0, 50% Ethanol 
                   
                   
               
               
                   
                 Chamber 4  
                 NH4 Bisulfite 
                 ~150  
                 μL 
               
               
                   
                 Chamber 5  
                 Tris pH 8.5 
                 2.0  
                 mL 
               
            
           
           
               
               
               
               
            
               
                   
                 Chamber 6  
                 Empty 
                 N/A 
               
               
                   
                 Chamber 7  
                 Empty 
                 N/A 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Chamber 8  
                 PEG200 
                 1.0  
                 mL 
               
            
           
           
               
               
               
               
            
               
                   
                 Chamber 9  
                 Empty 
                 N/A 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Chamber 10 
                 15 mM KOH 
                 500  
                 μL 
               
            
           
           
               
               
               
               
            
               
                   
                 Chamber 11 
                 Empty 
                 N/A 
               
               
                   
               
            
           
         
       
     
     In certain, illustrative, but non-limiting embodiments, the second cartridge is configured as shown in Table 10. In certain embodiments the chamber numbering corresponds to the chambers illustrated in  FIG. 1B . 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 One illustrative, but non-limiting configuration of a 
               
               
                 ″second″ cartridge for desulphonation of converted DNA 
               
               
                 and optional PCR of the desulphonated product. 
               
            
           
           
               
               
               
            
               
                 Chamber # 
                 Reagent 
                 Volume 
               
               
                   
               
               
                 Chamber 1  
                 Empty 
                 N/A 
               
               
                 Chamber 2  
                 1.25M GTC, 25 mM Tris 
                 2.0 mL 
               
               
                   
                 pH 7.0, 50% Ethanol 
                   
               
               
                 Chamber 3 with  
                 Sample from 1st  
                 ~1.6 mL 
               
               
                 funnel 
                 Cartridge 
                   
               
               
                 Chamber 4  
                 Empty 
                 N/A 
               
               
                 Chamber 5  
                 Tris pH 8.5 
                 2.0 mL 
               
               
                 Chamber 6  
                 Empty 
                 N/A 
               
               
                 Chamber 7  
                 Empty 
                 N/A 
               
               
                 Chamber 8  
                 PEG200 
                 1.0 mL 
               
               
                 Chamber 9  
                 Enzyme Bead, 1×  
                 2× Beads 
               
               
                   
                 Internal Bead 
                   
               
               
                 Chamber 10 
                 15 mM KOH 
                 500 μL 
               
               
                 Chamber 11 
                 Tris Bead, Enzyme Bead, 
                 3× Beads 
               
               
                   
                 1× External Bead 
               
               
                   
               
            
           
         
       
     
     In certain embodiments the second cartridge is configured to use one or more of the following genes as control genes: MYOD1, COL2A1, NONO, and/or TUBB. 
     In certain embodiments using the two-cartridge format is possible, inter alia, to run a non-nested PCR reaction or a methylation specific preamp in the 2 nd  cartridge. 
     The foregoing configurations are illustrative and non-limiting. Using the teaching provided herein numerous other two-cartridge formats will be available to one of skill in the art. 
     By way of illustration, data showing the results of a two-cartridge analysis of urine and sputum samples are shown in  FIGS. 35, 36, and 37 . In particular,  FIG. 35  shows a two-cartridge methylation analysis of BNC1 and ACTB. 0, 25, 50, and 100 copies of 100% methylated control DNA was spiked into serum and processed in the 1st sample prep cartridge (see, e.g., Table 9) , and then qPCR was run on the extracted, converted DNA sample in the second cartridge (see, e.g., Table 10) containing primer and probe sets for the methylated BNC1 promoter and a methylation independent primer and probe set for the ACTB promoter. 
       FIG. 36  shows the results of bisulfite conversion analysis of normal urine samples. 2.0 mL of normal urine was mixed with 1.5 mL of Lysis Buffer [4.5M GTC, 1% Tween20] and 0.5 mL of Ethanol in Condition #6. 1.5 mL of normal urine was mixed with 1.25 mL of Lysis Buffer and 1.25 mL of Ethanol in Condition #7. The 4.0 mL samples were pipetted into Chamber 2 of the methylation analysis cartridge 2 and analyzed for converted ACTB (right) and unconverted HMBS (left). 
       FIG. 37  shows the results of methylation analysis of normal and cancer sputum samples. Seven sputum samples were tested for SOX17, TAC1, and HOXA7 methylation using the GENEXPERT® methylation cartridge (C) and a comparative assay (M). Both raw Ct&#39;s and a deltaCt (to ACTB) are reported in this figure. 
     The foregoing configurations are illustrative and non-limiting. Using the teaching provided herein numerous other two-cartridge formats will be available to one of skill in the art. 
     cfDNA Sample Preparation Cartridge. 
     In certain embodiments a sample preparation cartridge is provided that is articular well suited to the preparation (and optional analysis) of nucleic acids from plasma or serum is provide. One illustrative, but non-limiting embodiment is shown in  FIG. 17 . As illustrated therein in certain embodiments the cartridge comprises a channel or chamber comprising an affinity matrix that binds DNA, a plurality of chambers disposed around a central valve assembly and selectively in fluid communication with the central valve assembly where the central valve assembly is configured to accommodate a plunger that is capable of drawing fluid into or out of a chamber in fluid communication with the central valve where the plurality of chambers comprises: a chamber configured to receive up to about 5 ml or up to about 4 ml of sample solution; a chamber containing PEG (e.g., PEG200); a chamber containing GTC-EtOH; a chamber containing an alkaline solution (e.g., KOH); and a chamber containing a buffer (e.g., Tris). In certain embodiments the plurality of chambers further comprises a chamber containing a conversion reagent (e.g., a bisulfite reagent). In certain embodiments the plurality of chambers comprises a chamber containing a wash solution (e.g., GTC-ethanol wash (typically 1.25M guanidinium thiocyanate, 25 mM Tris pH 7.0, 50% ethanol)). In certain embodiments the plurality of chambers comprises a chamber containing beads comprising one or more PCR primers and/or probes. In certain embodiments the chamber containing PEG contains about 1 ml of PEG. In certain embodiments the chamber containing an alkaline solution contains about 500 μL of solution. In certain embodiments the chamber containing GTC-EtOH contains about 2 ml GTC-EtOH. In certain embodiments the chamber containing a buffer contains about 2 mL of buffer. 
     It will be recognized that this configuration is illustrative, and using the teaching provided herein numerous other preparation cartridge configurations will be available to one of skill in the art. 
     Use of DABSO as an Alternative to Bisulfite 
     It was a surprising discovery that DABSO can be used to perform a conversion of DNA in a manner analogous to the use of bisulfites for the conversion of DNA and detection of methylation. Accordingly, in certain embodiments, methods of utilizing DABSO to converting cytosine residues in a DNA to uracil, while leaving 5-methylcytosine residues substantially unaffected are provided. In certain embodiments the methods involve contacting a sample comprising DNA with DABSO to convert the DNA, and desulphonating the converted DNA, to produce a DNA in which cytosine residues are converted to uracil, but 5-methylcytosine residues substantially unaffected. In certain embodiments the DABSO is provided at a concentration ranging from about 2 M up to about 5 M. In certain embodiments the DABSO is provided at a concentration of about 2.5 M. In certain embodiments the DABSO is dissolved in an alkaline aqueous solution (e.g., a KOH solution). In certain embodiments the reagent comprising DABSO comprises DABSO dissolved in a solution comprising KOH. 
     In certain embodiments the methods involve heating the DABSO/DNA solution to a temperature ranging from about 55° C. to about 90° C. In certain embodiments the DABSO is reacted with the DNA for a period of time ranging from about 15 minutes up to about 90 minutes. After the DNA is converted, it is desulphonated (e.g., by contacting the converted DNA with an alkaline reagent (e.g., KOH solution). In certain embodiments the conversion and/or desulphonation is performed on the DNA bound to a column, while in other embodiments the conversion and/or desulphonation is performed on the DNA in solution. 
     Also provided are methods of analyzing DNA methylation, where the methods involve providing a DNA sample, converting DNA in the sample using a DABSO reagent, e.g., as described above, and performing methylation specific PCR and/or nucleic acid sequencing, and/or high resolution melting analysis (HRM) on the converted nucleic acid to determine the methylation of said nucleic acid. In certain embodiments the providing of a DNA sample comprises preparing a sample as described herein (e.g., using lysis solutions and/or preparation cartridges as described herein. 
     Kits. 
     Kits for Methylation Detection. 
     In certain embodiments kits are provided for performing the methods described herein. In one illustrative embodiment, the kits comprise a container containing a reaction cartridge as described herein, a container containing a sample processing reagent as described herein, and a container containing a conversion reagent (e.g., a bisulfite reagent) as described herein. In certain embodiments the bisulfite reagent is provided in a chamber of the cartridge. In certain embodiments the bisulfite reagent is provided in a container separate from the cartridge. In certain embodiments, the sample processing reagent is provided in a chamber of the cartridge. In certain embodiments, particularly where the sample processing reagent comprises guanidinium thiocyanate the sample processing reagent is provided in a container separate from the cartridge. 
     In certain embodiments the kits can contain the cartridges for a two-cartridge set as described herein. Thus, in certain embodiments the kits can contain a “first” cartridge that is a sample preparation cartridge configured to prepare a sample and perform a bisulfite conversion of DNA in the sample (see, e.g.,  FIG. 20B , left panel) and a “second” cartridge configured to desulphonated the converted DNA and, in certain embodiments, perform a subsequent analysis (e.g., methylation-specific PCR) (see, e.g.,  FIG. 20B , right panel). In certain embodiments the first cartridge and the second cartridge are provided in separate containers in the kit, while in other embodiments, the first cartridge and second cartridge are provided in the same container. In certain embodiments the two-cartridge kits can contain devices (e.g., funnels, pipette tips, etc.) to facilitate transfer of a sample from the sample removal chamber of the first cartridge to the sample receiving chamber of the second cartridge. 
     In addition, the kits optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the use of the cartridges described herein to determine DNA methylation and, optionally, RNA expression. 
     In certain embodiments a kit for the determination of DNA methylation is provided where the kit comprises a container containing a cartridge for determining the methylation state of a nucleic acid as described herein. In certain embodiments the kit further comprises a lysis solution as described herein (e.g., a lysis solution for serum or plasma, e.g., as described in Table 13, and/or a lysis solution for FFPE samples, e.g., as described in Table 14). In certain embodiments the kit comprises a container containing proteinase K. In certain embodiments the kit contains a conversion reagent (e.g., a bisulfite reagent) in the cartridge or in a container separate from the cartridge. In certain embodiments the separate container can contain a pre-measured volume of conversion reagent suitable for one “run” of the cartridge. In certain embodiments the conversion reagent comprises a compound selected from the group consisting of sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, and DABSO. In certain embodiments the kit comprises a container containing a sample processing reagent. In certain embodiments the sample processing reagent comprises guanidium thiocyanate and/or ethanol. 
     In various embodiments the kit can additionally contain a cartridge for sample preparation as described herein (e.g., as illustrated in  FIG. 20 ). 
     In certain embodiments the kit contains instructional materials teaching the use of a cartridge for the determination of DNA methylation. Where a sample preparation cartridge is included in the kit the kit can additionally contain instructional materials teaching the use and operation of the sample preparation cartridge. 
     Kits for DABSO DNA Conversion and Methylation Detection. 
     In certain embodiments kits are provided for the use of DABSO as a conversion reagent, e.g., in the detection of the methylation state of a DNA. In certain embodiments the kits comprise a container containing a conversion reagent comprising DABSO, and a container containing a desulphonation reagent. In certain embodiments the kit comprises a column comprising an affinity matrix (e.g., a silica matrix material). In certain embodiments the kits comprise a container containing a binding buffer and/or a container containing an elution buffer. In certain embodiments the kit comprises a container containing a wash buffer. 
     In certain embodiments the kit further comprises a lysis solution as described herein (e.g., a lysis solution for serum or plasma, e.g., as described in Table 13, and/or a lysis solution for FFPE samples, e.g., as described in Table 14). In certain embodiments the kit comprises a container containing proteinase K. 
     In various embodiments the kit can additionally contain a cartridge for sample preparation as described herein (e.g., as illustrated in  FIG. 20 ). 
     In certain embodiments the kit contains instructional materials teaching the use of the kit to convert a nuclei acid for determination of the methylation state of the nucleic acid. 
     While the instructional materials in the kits described above typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. 
     EXAMPLES 
     The following examples are offered to illustrate, but not to limit the claimed invention. 
     Example 1 
     To validate the method human genomic DNA (HGDNA) was used as a starting sample to monitor sample preparation, bisulfate conversion, sample cleanup, and methylation specific qPCR in a Cepheid GENEXPERT® cartridge. In order to measure bisulfite conversion efficiency, half of the DNA-bisulfite mix was loaded and heated in the 50 μL cartridge tube during the bisulfite conversion step. Therefore, under optimal conversion conditions approximately half of the HGDNA is converted and the other half remains unconverted. 
     Primers and Taqman probes for the qPCR step were designed for one unconverted gene (HMBS (hydroxymethylbilane synthase housekeeping gene)) and one converted gene (ACTB (beta actin)), and the conversion efficiency was then quantitated by comparison of cycle threshold values (Cts). Both ACTB and HMBS are commonly used as single or low copy reference genes, and thus we expect similar copy numbers per ng of HGDNA. 
     A representative GENEXPERT® run from 300 ng of HGDNA is shown below in  FIG. 5 , with the ACTB qPCR curve in green and the HMBS qPCR curve in blue. The qPCR reaction was run for 45 cycles with a 3 temperature cycle of 96° C. for 5 seconds, 60° C. for 15 seconds, and 72° C. for 15 seconds. At a manual threshold setting of 20 fluorescence units we observed a Ct of 31.7 for the converted ACTB gene and a Ct of 32.7 for the unconverted HMBS gene. Importantly, this result demonstrates that we are able to achieve near-optimal bisulfite conversion efficiency of HGDNA in our cartridge at physiological relevant concentrations of DNA found in FFPE tissue slices and plasma/serum samples. Further specificity for fully converted sequences can be achieved through a nested qPCR reaction or by heating the entire sample. However, neither option would be absolutely required for methylation specific qPCR in the GENEXPERT® because primer and probe sets are designed to amplify only the converted sequences. Thus remaining unconverted DNA sequences would act as carrier DNA, which notably is frequently added during bisulfite conversion, DNA isolation, and PCR methods. 
     Example 2 
       FIGS. 6A and 6B  show the linearity of converted ACTB. In particular,  FIG. 6A  shows the results of a 15 cycle nested qPCR for ACTB using hgDNA. As can be seen from the panel on the right the signal (Ct value) is substantially linear between about 25,000 copies and about 100 copies.  FIG. 6B  shows the results of a 20 cycle nested qPCR for ACTB using hgDNA. These plots demonstrate the sensitivity of the cartridge for hgDNA . Dropouts start occurring around 20-50 copies with a sensitivity of about 25 copies of converted DNA. 
       FIGS. 7A, 7B, and 7C  show the results of qPCR for six methylated targets (AKR1B1, HOXB4, TM6SF1, RASGRF2, and RASSF1A).  FIG. 7A  show the results of 20 cycle nested qPCR for controls (25 ng of HSDNA, and 5000 MBA-453 cells whose DNA is not bisulfite-converted).  FIG. 7B  shows the results of 20 cycle nested qPCR for the six methylated targets using DNA from MBA-453 cells that has been bisulfite converted. A strong signal is shown for all targets. HIST1H3C was not reliably detected.  FIG. 7C  shows the results of 20 cycle nested qPCR for the six methylated targets using DNA from MBA-453 cells that has been bisulfite converted and is in a carrier comprising 1 μg of SS and 10 ng of HS DNA. Dropouts were observed at about 100 cells and below, however, with the carrier, there were significantly fewer dropouts. 
     Example 3 
       FIG. 8  illustrates the results of a determination of conversion efficiency. The conversion efficiency is about 66% (˜1 Ct) the difference between unconverted HMBS and converted ACTB. Ideal Ct with 100% binding/elution, 100% conversion, and 100% binding elution is about 24-25. The experiments appear to show a 50% binding/elution, 50-66% conversion, and 50% binding/elution for a 10-fold reduction and a Ct of about 27. 
       FIG. 9  illustrates the increase in specificity for converted DNA produced by nested qPCR. Nested PCR appears to increase the specificity for converted DNA, to increase the specificity for methylated DNA and to reduce contamination issues. 
       FIG. 10  illustrates the specificity of the methylation cartridge. No specificity is shown for unconverted DNA (top panel) or unmethylated DNA (bottom panel) except for HIST1H3C. 
       FIGS. 11A and 11B  show some illustrative but non-limiting workflows for analysis of methylation using a cartridge (e.g., a GENEXPERT® cartridge).  FIG. 11A  illustrates one work flow for analysis of DNA methylation in a serum sample. As illustrated in this workflow, serum is added to a lysis reagent vial and mixed/vortexed. The sample is then dispensed into a sample port in the cartridge. The cartridge is placed in the system for analysis. 
       FIG. 11A  illustrates one work flow for analysis of DNA methylation in a tissue section (e.g., frozen or formalin-fixed paraffin embedded (FFPE) section). As shown therein, in one embodiment, a tissue section (e.g., a 4 um FFPE section) is provided. FFPE lysis reagents are added (see, e.g., PCT/US2013/061863 (WO/2014/052551 for illustrative lysis reagents) and the mixture can be heated. Ethanol can be added and the mixture vortexed. The sample is then dispensed into a sample port in the cartridge. The cartridge is placed in the system for analysis. 
       FIG. 12  illustrates the results for a FFPE cell button for converted ALU and methylated RASSF1A. 
     Example 4 
     Detection of Markers for Breast Cancer Monitoring 
     Materials and Methods: 
     Either 1000 MBA-453 cells or 25 ng of human sperm (HS) DNA were added to 2.5 mL of binding buffer (2.25 M Guanidinium thiocyanate, 22.5 mM Tris pH 7.0, 0.5% Tween20, 50% Ethanol, and 0.005% SE-15 antifoam). The 2.5 mL solution of cells or DNA was added to chamber 2 of the Cepheid methylation cartridge (layout in  FIG. 13A ). The remaining chambers in the methylation cartridge were filled as follows: Chamber 3—3.2 mL of Wash buffer (1.25M Guanidinium thiocyanate, 25 mM Tris pH 7.0, 50% Ethanol), Chamber 4—90 μL of 7M Ammonium Bisulfite, Chamber 5—4 mL of 50 mM Tris pH 8.5, Chamber 8—1 mL of PEG200 Rinse, Chamber 9—quantitative PCR beads including EZR (Taq) and TSR (6 target breast cancer multiplex for RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1, see Table 11, below), Chamber 10—500 μL of 15 mM KOH, and Chamber 11—nested beads including EZR (Taq) and TSR (6 target breast cancer multiplex for RASSF1A, AKR1B1, HOXB4, HIST1H3C, RASGRF2, TM6SF1). The methylation cartridge was then loaded into a Cepheid GeneXpert and the entirety of the methylation assay was completed by the GeneXpert—the first DNA sample prep, the bisulfate conversion, the second post conversion DNA sample prep, the desulphonation, and the 20 cycle nested and quantitative PCR reactions. 
     A flow chart illustrating the methylation protocol is shown in  FIG. 13B . It is noted that the PEG200 was filled in the waste chamber 8, and after the assay starts the PEG200 is dispensed into Chamber 1. The PEG200 is a viscous liquid that cannot easily be directly loaded in the smaller chamber 1. Additionally, chamber 1 acts as an air chamber when the cartridge is first loaded before becoming the PEG200 chamber. Thus, the assay begins with Chamber 1=air and Chamber 8=PEG200 and is quickly switched to Chamber 1=PEG200 and Chamber 8=Waste after cartridge loading. 
     The numbers shown in the “Initial Vol.” column of  FIG. 13A  just refer to liquid volumes. In this case there are just 2× beads in chamber 11—1× TSR bead (primer and probes for the 6 targets) and 1× EZR bead (Phoenix Taq). These beads are for the final qPCR reaction. Similarly, there are 3× beads in chamber 9—1× TSR bead (primers for the 6 targets), 1× Tris bead (to quench KOH) and 1× EZR bead (Phoenix Taq). These beads are for the first 15-20 cycle PCR reaction. 
     It is also noted that Chamber 6 is an air chamber throughout the entire assay and is never filled. Chamber 7 is used as sort of a gateway to the PCR tube in the back of the cartridge. It is not filled to start the assay but is filled during the assay on 3 occasions before loading into the tube—1) the DNA-bisulfite mix that is heated in the tube for conversion 2) the 15-20 cycle PCR reaction and 3) the final qPCR reaction. 
     The primers shown in the Table 11 provided shows five sequences for each gene—two extension primers and 2 qPCR primers for each nested amplification and one probe. The first 15-20 cycle PCR reaction was not specific for methylation but only the converted DNA sequences (i.e., they do not cross CpGs and in a couple instances when they do we use an R=purine or Y=pyrimidine to catch both methylated and unmethylated). The second 45 cycle qPCR reaction contains both primers and probes that are specific for typically 2-3 methylated CpGs. 
     Results: 
     The methylation cartridge was run using 1000 MBA-453 cells with and without bisulfite ( FIG. 15A-15B ) and 25 ng of HS DNA with bisulfite ( FIG. 15C ) that was primarily unmethylated at each gene promoter with the exception of HIST1H3C. There was little or no amplification of any of the targets in either the no bisulfite or unmethylated HS DNA control reactions ( FIG. 15A, 15C ). With the addition of bisulfite, the methylation cartridge picked up high levels of methylation at multiple gene promoters from 1000 MBA-453 cells, specifically AKR1B1, RASSF1A, HOXB4, and RASGRF2. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Nested primers for R4SSF1A, AKR1B1, HOXB4, HIST1H3C, 
               
               
                 RASGRF2, and TM6SF1. C*, T* are optionally functionalized 
               
               
                 (e.g., to alter probe Tm) bases. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                 Gene/ 
                   
                   
                 ID 
               
               
                 Probe name 
                 Type 
                 SEQUENCE 
                 NO 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 RASSF1A 
                   
                   
                   
               
               
                 olAK61 
                 ext 
                 GTTTTATAGTT(T*)TTGTATTTAGG 
                 62 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK41 
                 ext 
                 AACTCAATAAACTCAAACTCCC 
                 42 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK1 
                 qPCR 
                 GCGTTGAAGTCGGGGTTC 
                 2 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK2 
                 qPCR 
                 CCCGTACTTCGCTAACTTTAAACG 
                 3 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK63 
                 qPCR 
                 fluor-(C*)TGGTTTCGT(T- 
                 64 
               
               
                   
                 probe 
                 quencher)CGGT(T*)CGCG-quencher/blocker 
                   
               
               
                   
               
               
                 HIST1H3C 
                   
                   
                   
               
               
                 olAK60 
                 ext 
                 GGATTTTTGAAATATTATAGGATTAATTAG 
                 61 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK43 
                 ext 
                 ATAAAATTTCTTCACRCCACC 
                 44 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK59 
                 qPCR 
                 TCGTACGAAGTAAATAGTTCGTAAG 
                 60 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK54 
                 qPCR 
                 CCGATAACCGAAACGCTCTTAC 
                 55 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK65 
                 qPCR 
                 fluor- 
                 66 
               
               
                   
                 probe 
                 C AAACTACTTACGCGAAACTT(T*)ACCGCC- 
                   
               
               
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 RASGRF2 
                   
                   
                   
               
               
                 olAK44 
                 ext 
                 GAGGGAGTTAGTTGGGTTAT 
                 45 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK45 
                 ext 
                 CCTCCAAAAAATACATACCC 
                 46 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK13 
                 qPCR 
                 GTAAGAAGACGGTCGAGGCG 
                 14 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK14 
                 qPCR 
                 ACAACTCTACTCGCCCTCGAA 
                 15 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK67 
                 qPCR 
                 fluor- 
                 129 
               
               
                   
                 probe 
                 AAACGAACCACTTCTCG(T*)ACCAACGAC- 
                   
               
               
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 AKR1B1 
                   
                   
                   
               
               
                 olAK58 
                 ext 
                 GYGTAATTAAT(T*)AGAAGGTTTTTT 
                 59 
               
               
                 olAK47 
                 ext 
                 AACACCTACCTTCCAAATAC 
                 48 
               
               
                 olAK19 
                 qPCR 
                 GC GCGTTAATCGTAGGCGTTT 
                 20 
               
               
                 olAK20 
                 qPCR 
                 CCCAATACGATACGACCTTAAC 
                 21 
               
               
                 olAK75 
                 qPCR 
                 fluor-(C*)A(C*)GCGTACCT(T- 
                 76 
               
               
                   
                   
                 quencher)TAAA(T*)AAC CC G(T*)AAAATCG- 
                   
               
               
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 HOXB4 
                   
                   
                   
               
               
                 olAK48 
                 ext 
                 TTAGAGGYGAGAGAGTAGTT 
                 49 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK49 
                 ext 
                 AAACTACTACTAACCRCCTC 
                 50 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK25 
                 qPCR 
                 CGGGATTTTGGGTTTTCGTCG 
                 26 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK26 
                 qPCR 
                 CGACGAATAACGACGCAAAAAC 
                 27 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK76 
                 qPCR 
                 fluor- 
                 77 
               
               
                   
                 probe 
                 AACCGAACGA(T*)AACGAAA(N*)ACGACGAA- 
                   
               
               
                   
                   
                 quencher/blocker 
                   
               
               
                   
               
               
                 TM6SF1 
                   
                   
                   
               
               
                 olAK50 
                 ext 
                 AGGAGATATYGTTGAGGGGA 
                 51 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK51 
                 ext 
                 TCACTCATACTAAACCRCCAA 
                 52 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK56 
                 qPCR 
                 GTTTAGCGGGATGCGGTG 
                 57 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK57 
                 qPCR 
                 ACACGAAAACCCCGATAAC 
                 58 
               
               
                   
                 primer 
                   
                   
               
               
                 olAK77 
                 qPCR 
                 fluor-AAACACTCATCGCAACCGCCGCG- 
                 34 
               
               
                   
                 probe 
                 quencher/blocker 
               
               
                   
               
            
           
         
       
     
     The primers shown in Table 11 are illustrative and not limiting. Numerous other primers and nested primer sets will be available to those of skill in the art. By way of example, illustrative primers for the detection of methylation of ADAMTS1 and BNC1 genes associated with pancreatic cancer and for the detection of methylation of the MGMT gene associated with glioma are shown in Table 12. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Illustrative primers for the detection of 
               
               
                 methylation of ADAMTS1 and BNC1 genes associated 
               
               
                 with pancreatic cancer and for the detection of 
               
               
                 methylation of the MGMT gene associated with 
               
               
                 glioma. 
               
            
           
           
               
               
               
               
            
               
                 Gene/ 
                   
                   
                 SEQ 
               
               
                 Probe 
                   
                   
                 ID 
               
               
                 name 
                 Type 
                 SEQUENCE 
                 NO 
               
               
                   
               
               
                 BNC1 
                 ext 
                 CCCRCAAACCRCGAAAACCTC 
                 227 
               
               
                   
                 ext 
                 CCGACGACCGACG 
                 235 
               
               
                   
                 qPCR 
                 GTTTTTTTTYGGGAGAGGTAAATA 
                 228 
               
               
                   
                 qPCR 
                 GGGAGAGGTAAATATCGATAC 
                 236 
               
               
                   
                 qPCR 
                 fluor-TGGYGGGGG(T*)AGA(T*) 
                   
               
               
                   
                   
                 ATTTT-quencher/blocker 
                 389 
               
               
                   
               
               
                 ADAMTS1 
                 ext 
                 CRCCTCCRAAACTAAAACAAC 
                 229 
               
               
                   
                 ext 
                 CGCGAAAATTAATACCTAACG 
                 237 
               
               
                   
                 qPCR 
                 GGGTTATTGTAAAGTTAGGGTG 
                 230 
               
               
                   
                 qPCR 
                 TTAGGGTGCGTTATCGGAC 
                 238 
               
               
                   
                 qPCR 
                 fluor-TCTACTCAAAACTCTCCCCTCT 
                   
               
               
                   
                   
                 CC-quencher/blocker 
                 390 
               
               
                   
               
               
                 MGMT 
                 ext1 
                 GTTTT(T*)AGAAYG(T*)TTTGYGTTT 
                 263 
               
               
                   
                 ext2 
                 AAAAAAC(T*)CCRCACTCTTCC 
                 265 
               
               
                   
                 qPCR 
                 TTTCGACGTTCGTAGGTTTTCGC 
                 266 
               
               
                   
                 qPCR 
                 GCACTCTTCCGAAAACGAAACG 
                 267 
               
               
                   
                 qPCR 
                 fluor- 
                 268 
               
               
                   
                 probe 
                 CCAAACAC(T*)CACCAAATC(N*)CAA 
                   
               
               
                   
                   
                 AC-quencher/blocker 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Sample Preparation for Plasma and FFPE Samples 
       FIG. 17  illustrates one configuration of a cartridge that can be used to prepare DNA samples for PCR and/or methylation detection. The sample, obtained from serum or plasma, or an FFPE sample can simply be introduced into a sample chamber of the cartridge (e.g., chamber 3) and operation of the cartridge as described herein provides a sample ready for PCR and/or methylation detection. 
     Sample Preparation 
     In one illustrative, but non limiting embodiment, a serum or plasma sample is prepared (e.g., for analysis of cfDNA) by treating the serum or plasma with proteinase K. Then the proteinase K treated serum/plasma is mixed with a lysis solution comprising guanidinium thiocyanate (GTC), buffer (e.g., Tris pH 7.0), a detergent (e.g., Tween 20), and an optional antifoam (e.g., antifoam SE15). An alcohol (e.g., isopropanol) is added to the solution which is then introduced into the cartridge for sample processing. In one embodiment the lysis solution is formulated as shown in Table 13. The proteinase K treated serum/plasma can be mixed with lysis solution and alcohol in a ratio corresponding to 1.3 mL proteinase K treated serum/plasma, 2.2 mL lysis solution, and 1.5 ml alcohol. In certain embodiments the serum/plasma sample is treated with proteinase K for about 15 minutes. The lysis solution is added cold and held/mixed for about 10 minutes. Then isopropanol is added to the mixture which is then loaded into the cartridge for processing. 
     As noted above, for serum/plasma the alcohol (e.g., isopropanol) precipitations are typically done at RT, and in particular typically not performed with “salty” solutions. In certain embodiments longer room temperature precipitation times can be used. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Lysis solution for serum or plasma. 
               
            
           
           
               
               
               
            
               
                   
                 Reagent 
                 Amount 
               
               
                   
                   
               
               
                   
                 Guanidine thiocyanate (GTC) 
                 4.5M 
               
               
                   
                 Buffer (e.g., Tris) pH 7.0 
                 45 mM 
               
               
                   
                 Detergent (e.g., Tween20) 
                    1% 
               
               
                   
                 Antifoam SE15 
                 0.01% 
               
               
                   
                   
               
            
           
         
       
     
     In another illustrative, but non-limiting embodiment, a formalin fixed paraffin-embedded (FFPE) sample is prepared by combining the FFPE sample with proteinase K and a lysis solution comprising a buffer (e.g., HEPES), a chelator (e.g., EDTA), NaCl, MgCl 2 , and optionally sodium azide and/or an antifoaming agent. The solution is heated (e.g., at 70° C. to 90° C.) for a period of time ranging, for example from about 10 minutes up to about 4 hours. An alcohol is added to the solution and the solution is then introduced into the cartridge for sample processing. In one embodiment the lysis solution is formulated as shown in Table 14. In one illustrative, but non-limiting embodiment, 1.2 mL of the lysis solution shown in Table 14 is added to the FFPE section(s). Proteinase K is added and the mixture is heated, e.g. at 80° C. for about 15 minutes. In certain embodiments heating is performed at 56° C. for 2 hours followed by 90° C. for 30 minutes. Then 1.2 mL of ethanol is added to the mixture and the mixture is loaded into a sample chamber of the cartridge for processing. 
     
       
         
           
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 Lysis buffer for formalin fixed paraffin embedded (FFPE) sample. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Tween20 
                    1% 
               
               
                   
                 NaCl 
                 400 mM 
               
               
                   
                 EDTA 
                  25 mM 
               
               
                   
                 MgCl 2   
                  10 mM 
               
               
                   
                 HEPES pH 7.2 
                  50 mM 
               
               
                   
                 Sodium Azide 
                 0.01% 
               
               
                   
                 SE15 
                 0.01% 
               
               
                   
                   
               
            
           
         
       
     
     Cartridge Operation and Extraction Performance. 
     When cfDNA is being prepared, in certain embodiments, it is possible to include extraction controls to permit monitoring of the quality of the DNA preparation. As illustrated in  FIG. 18 , there are two different bead sets. One bead set contains an endogenous HMBS primer and probe set for a SAC (sample assay control) and exogenous BG primer and probe set for a SPC (sample prep control). The other contains an endogenous Beta-Globin PP set for SAC (as well as BG SPC). 
     It was discovered, inter alia, that the use of GTC in the cartridge may be less important for serum than plasma samples. Without being bound by a particular theory it is believed that this may be due to the fact that serum contains less protein. Accordingly, in certain embodiments, the cartridge may contain less GTC or may omit GTC. 
       FIGS. 19A and 19B  show a comparison of the results of cfDNA preparation performed using a cartridge as described herein compared to the results obtained using a conventional “tubefill” procedure. As illustrated in the qPCR results shown in  FIG. 19A , the binding and elution efficiencies obtained using the cartridge are extremely close (within one Ct) to those obtained using the tubefill protocol. As illustrated in  FIG. 19B  titrations of sample concentrations show that the cartridge preparation is conservatively within 1 Ct of the tubefill preparation down to a sample concentration as low as about 10 pg. It is believe the cartridge preparation is even closer to the tubefill protocol at higher sample concentrations. 
     Example 6 
     Testing a High-Volume Sample Preparation Cartridge 
     In certain embodiments high volume sample preparation (HSVP) cartridges are provided for the preparation of large volumes of sample (e.g., up to about 12 ml to 15 ml). This is particularly useful where the sample contains DNA at a low concentration (e.g., cfDNA in serum or plasma). One such cartridge is schematically illustrated in  FIG. 20A . As shown therein the cartridge provides three chambers (chambers 2, 3, and 5) that can be used to receive a sample. In the illustrated embodiment, each of these chambers can receive about 4 mL of sample and, in certain embodiments, the sample comprises 4 mL of plasma/serum combined with 4 mL of GTC and 4 mL of alcohol (e.g., isopropanol). 
     The sample is introduced into these chambers and the cartridge is operated as described herein to prepare the sample for PCR and/or methylation analysis. By way of illustration, in certain embodiments, operation of this cartridge can comprise binding DNA to an affinity column (e.g., for cleanup) and eluting the DNA. In certain embodiments where a methylation analysis is to be performed, the operation of the cartridge can further comprise combining the DNA with a conversion reagent (e.g., a bisulfate as described herein) and heating the mixture to convert the DNA. In certain embodiments, the HSVP cartridge can also be configured to desulphonates the converted DNA. In other embodiments, the DNA can be desulphonated in the second (e.g., qPCR) cartridge as schematically illustrated in  FIG. 20B . The second cartridge can also perform the methylation analysis (e.g. a qPCR analysis). 
       FIG. 21  shows a comparison of sample preparation results of DNA from plasma and serum between one cartridge and two cartridge protocols using the HMBS or β-globin primer and probe set. As shown therein, there was a linear increase in DNA recovery between 0.5 mL and 4 mL of serum or plasma. Moreover there was little to no loss when using one cartridge for the preparation and analysis or when using separate cartridges for preparation and analysis/ 
     Example 7 
     Optimizing Bisulfate Conversion 
     In certain embodiments when using a cartridge for a methylation analysis as described herein one potential issue is the optimization of elution efficiently using the smallest volume possible. Small elution volumes are easier to deal with using spin columns. This problem can be addressed by using multiple heating steps to process larger sample volumes. 
     A second technical concern arises when heating a larger sample (e.g., minimum 100 μL) when using a smaller (e.g., 50 μL) heating tube or chamber. In certain instances, pressurizations between heating steps can make it difficult to reproducibly account for volume aspirates and dispenses. Secondly, the absence of pressurization can lead to to volume changes and bubbles especially at higher temperatures. Thirdly, it is possible to pick up air between heated and unheated samples during port changes in between heat steps. 
     To investigate these optimization of bisulfate conversion in a 50 μL tube using single and double heating steps was investigated. This experiment was performed as follows: 
     Pull 75-80 μL of bisulfate-DNA; heat 95° C.-10 s, 65° C.-300 s×8; 
     Pull rest+5-10 μL; pressurize; heat 95° C.-480 s, 65° C.-1800 s×1. 
     The results for 0.5 mL of serum are shown in  FIG. 22  where the top panel is 1× Heat (converted 33.0, unconverted 34.4) N=4, and the bottom panel is 2× Heat (converted 31.9, unconverted 36.1) N=4. 
     There is a gain of about 1 Ct in the converted ACTB signal when going from 1× heat to 2× heat. This suggests almost all of the DNA is converted. This is supported by the fact that there is also a loss of about 2 Ct&#39;s in the unconverted HMBS signal. A 1 Ct increase is logical since we went from heating 50/100 μL to 100/100 μL of DNA-bisulfite sample. 
     Example 8 
     Comparison of a DNA Methylation Cartridge with Tube-Based Commercial Kits 
       FIG. 23A  shows a comparison of the user steps required when performing a methylation analysis using cartridge as described herein (left) as compared to the steps required when using commercial kits (QIAamp MinElute Virus Spin Kit (Qiagen, Inc.), and EZ DNA Methylation-Lightning™ Kit (Zymo Research, Inc.)) to perform the same analysis. As can readily be seen the cartridge-based methylation analysis requires far few user steps with a labor time of about 5 minutes as compared to the 2-3 hour labor time required using the kits. 
     To compare the results produced by the different methods, 200 μL of serum was purified using the Qiagen kit. The DNA was converted using the Zymo kit, purified with a second spin column and eluted with 10 μL. Ran all 10 μL using converted unmethylated ACTB primers and probes (TSR). In comparison, 200 μL of serum were run in the methylation cartridge as described herein. Results are shown in  FIG. 23B . As is readily evident, the cartridge method produced results extremely comparable to those obtained using the commercial kits. However, this was accomplished with far less labor and time. 
     Example 9 
     Use of DABSO for DNA Conversion 
     It was initially attempted to dissolve 5 g DABSO in 5 mL H2O. Ultimately a few mLs of 10M KOH and a mL of water were added and heated to solubilize the DABSO and to raise the pH up to between about pH 5 and pH 5.5 at an estimated final DABSO concentration of ˜2.5M. 
       FIG. 24  shows graphs of tubefills of 750 ng of DNA converted using DABSO or the Zymo conversion reagent. The materials were offboard heated (1 μg) in a thermocycler and purified with spin columns and run as tubefills. The 3 different experiments were: 
     1) 120 uL DABSO/30 uL DNA; 
     2) 120 uL Zymo/30 uL DNA; and 
     3) 70 uL Zymo/30 uL DNA (ratio currently in the cartridge). 
     As shown in  FIG. 24 , DABSO provided good conversions almost comparable to those obtained using the Zymo reagent. 
     Example 10 
     Sensitivity of Detection of Methylated DNA 
     To evaluate the sensitivity of detection of DNA methylation, converted ACTB gene promoter was detected as a function of copy number using a cartridge as described herein. The goal was to detect less than 25 copies of converted, unmethylated DNA. As previously shown, fallouts were observed at about 10-50 copies (1 fallout each). Similar sensitivity was observed for methylated DNA targets in a serum background. 
       FIG. 25 , panel A, illustrates the detection of methylated DNA in a dilution series (MGMT (O-6-Methylguanine-DNA Methyltransferase gene)). As shown therein MGMT was detected down to a level of 78 pg. 
     The detection of methylated breast cancer markers RASSF1A and AKR1B1 in MBA-453 cells is shown in  FIG. 25 , panel B. As shown therein, breast cancer markers were detected down to 100 cells. 
     The detection of methylated pancreatic cancer markers ACTB, BNC1, and ADAMTS1 in a dilution series is shown in  FIG. 25 , panel C. As shown therein, pancreatic markers were detected down to 25 copies. 
     Table 15 shows the hit rate of pancreatic cancer markers BNC1 and ADAMTS1 as a function of concentration. As shown therein these markers could be detected below 120 pg. Note a positive “hit rate” is an amplification in either gene for a replicate. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 illustrates the hit rate for pancreatic marker  
               
               
                 detection as a function of concentration. 
               
            
           
           
               
               
               
            
               
                   
                 Concentration 
                 Hit rate (#/replicates) 
               
               
                   
                   
               
               
                   
                   0 pg 
                 0/3 
               
               
                   
                  16 pg 
                 6/8 
               
               
                   
                  30 pg 
                 5/8 
               
               
                   
                  60 pg 
                 4/8 
               
               
                   
                 120 pg 
                 4/4 
               
               
                   
                   
               
            
           
         
       
     
     Example 11 
     Reverse Complement Multiplex Assay for both Strands 
       FIG. 26  illustrates the results for a reverse complement multiplex assay for both DNA strands. Following bisulfite conversion, both strands lose their complementarity. Thus, primer and probe sets have to be designed for one strand or the other, and result in unique amplicons. In addition to providing “more opportunities”, this approach might potentially help with sensitivity (at LOD, if only one strand or the other ends up in the tube, this approach would ensure the signal gets picked up). 
     The multiplex assay allows the multiplex to detect different CpGs at the same promoter site. The reverse component multiplex provides more queries on target and the possibility to pick up heterogamous methylation. 
     Example 12 
     Detection of DNA Methylation and Mutation in a Single Cartridge 
     In certain embodiments the multiplex PCR reactions can contain primers and probes that permit the detection of mutations in addition to methylation in the same cartridge.  FIG. 27A  illustrates the detection of methylated BNC1 and ADAMTS1 along with the KRAS G12D mutation along with control BG (Top Panel) and the detection of methylated BNC1 and ADAMTS1 along with the KRAS wildtype along with control BG (Bottom Panel). 
       FIG. 27B  illustrates the simultaneous detection of BNC1 and ADAMTS1 methylation in PANC-1 cells (top panel) and MIA-PaCa cells (bottom panel) along with the KRAS G12D mutation. 
     Example 13 
     Multiplex Optimization of Pancreatic Cancer 
     It was determined that methylation analysis of ADAMTS1, BNC1, (and certain other genes) permits detection and/or staging of pancreatic cancer. Accordingly, the initial multiplex assay for BNC1 and ADAMTS1 was optimized to facilitate incorporation of probes for other genes. To optimize this assay temperature gradients were run on external and internal PCRs for forward/reverse bisulfite converted strands. Single-plexes (fwd/rev for each gene) were run at external temperatures of 56° C., 58° C., and 60° C. and internal temperatures of 64° C., 66° C., and 68° C. (see, e.g.,  FIG. 28 ). In certain embodiments the assays were developed as two 4-plexes for BNC1 and ADAMTS1 and two other genes, one 4-plex for methylation analysis of a forward strand and one 4-plex for methylation analysis of a reverse strand. 
     The probes were combined into two sets (see,  FIG. 29 ) based on preferred reaction conditions (salt conditions 40 mM (LS), 60 mM (MS), 80 mM (HS) KCl. 15 mM NH 4 SO 4 ) and optimized for specificity. The final optimized salt condition for multiplex 1 was 80 mM KCl, 5 mM MgCl 2 , 20 mM Tris pH 8.5, and 10 mM NH 4  and for multiplex 2 was 62 mM KCl, 4 mM MgCl 2 , 20 mM Tris pH 8.5, and 10 mM NH 4 . 
     Example 14 
     Detection of MGMT Methylation 
     The O(6)-methylguanine-DNA methyltransferase (MGMT) gene encodes a DNA repair enzyme that can abrogate the effects of alkylating chemotherapy such as temozolamide. If the MGMT gene is active, the damage is rapidly repaired. It is believed that malignant gliomas may have the MGMT gene inactivated due to methylation of its promoter region. Methylated MGMT gene is a predictive inicator for BETTER response to chemotherapy (as the tumor has no means to repair the DNA damage induced by the alkylating agent). 
     Primers and probes were developed for the detection of MGMT methylation as illustrated in  FIG. 30  and summarized below in Table 16. In particular,  FIG. 30  illustrates the converted template with CPGs (as determined from pyrosequencing) shown in grey. As illustrated after bisulfite conversion the forward and reverse strand are no longer complementary permitting separate analysis of each strand. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Illustrative primer/probe set for detection 
               
               
                 of MGMT methylation (see, e.g., FIG. 30). 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 Probe 
                   
                 ID 
               
               
                 Probe 
                   
                 Type 
                 Sequence 
                 NO 
               
               
                   
               
               
                 External 
                 22422 
                 MGMT 
                 GTTTT(T*)AGAAYG(T*) 
                 263 
               
               
                   
                   
                 Fwd-4 
                 TTTGYGTTT 
                   
               
               
                   
                 22423 
                 MGMT 
                 AAAAAAC(T*)CCRCACTC 
                 265 
               
               
                   
                   
                 Rev-4 
                 TTCC 
                   
               
               
                   
               
               
                 Internal 
                 22150 
                 MGMT 
                 TTTCGACGTTCGTAGGTTT 
                 266 
               
               
                   
                   
                 Fwd-2 
                 TCGC 
                   
               
               
                   
                 22151 
                 MGMT 
                 GCACTCTTCCGAAAACGAA 
                 267 
               
               
                   
                   
                 Rev-2 
                 ACG 
                   
               
               
                   
                 22419 
                 MGMT 
                 Fluor- 
                   
               
               
                   
                   
                 TaqMan-2 
                 CCAAACAC(T*)CACCAAA 
                 268 
               
               
                   
                   
                   
                 TC(N*)CAAAC 
               
               
                   
               
            
           
         
       
     
     To evaluate detection sensitivity a MGMT dilution series (5 ng to 78 pg MGMT DNA in a background of 20 ng of HS DNA)) was evaluated using ACTB as a control. In an illustrative experiment, 78 pg of methylated MGMT DNA was only about 10 cycles off the Ct of only unmethylated HS DNA. 
     As shown in  FIG. 31  results produced using the methylation cartridge described herein for the detection of MGMT methylation was compared to the results produced by pyrosequencing for extracted DNA ( FIG. 31 , top) and for a FFPE sample ( FIG. 31 , bottom). Pyrosequencing typically uses a cutoff between 10-15% to determine patient stratification. We used an arbitrary cutoff of 12.5 (between ACTB and MGMT) to match pyrosequencing results as closely as possible. Accordingly, in this example a cutoff was set at delta Ct=12.5 and calculated concordance with &gt;15% methylation. The cartridge analysis of the extracted DNA show a sensitivity of 90% and a specificity at 86% while the cartridge analysis of the FFPE sample showed a sensitivity of 88% and a specificity of 95%. 
     It is noted that specificity can be improved in two ways: 1) the annealing temperature can be increased as the 62° C. annealing temperature was rather low. Additionally methylation probes that cover 3 (or more) CpGs can be utilized. 
     Example 15 
     Detection of BRCA1 Methylation 
     BRCA1 is a caretaker gene responsible for repairing DNA. It is believe that BRCA1 is involved in homologous, recombination, non-homologous end joining, and nucleotide excision repair. Women with an abnormal BRCA1 gene have an 80% chance of developing breast cancer. 
     Without being bound to a particular theory, it is believed that BRCA1 methylation is a potential predictive marker of response to chemotherapy in triple negative BC patients. Study of NSCLC patient&#39;s treated with cisplatin showed those with low BRCA1 expression had improved survival rates. High levels reduced the effectiveness of chemotherapy by repairing the damage caused to cancer cells. 
     In view of these, and other, observations cartridges and methods of use were developed for detection of BRCA1 methylation. In particular, the PCR condition were optimized as follows: 1) External temperature was evaluated between 56-62° C. and we settled on a 3 step 56° C. annealing PCR protocol; 2) Internal temperature was evaluated between 64° C-70° C. and we settled on a two-step 68° C. annealing PCR protocol. Results are shown in  FIG. 32 . 
     For BRCA1, a one target assay was tested with the ACTB control gene. Eight different cell lines were tested and the effect of adding NH4 was compared (see,  FIG. 33 ). BRCA1 methylation was expected to be observed in the 3199 cell line. 
     Example 16 
     Detection of Gene Methylation Associated with Lung Cancer 
     A three target methylation assay for genes whose methylation is associated with lung cancer (SOX17, CD01, TAC1) was tested along with the ACTB control gene. The data shown in  FIG. 34  indicate that, as expected, the 3 targets do not come up in a background of normal plasma but are present to some degree in three different lung cancer cell lines. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.