Patent Publication Number: US-2015080235-A1

Title: Assays and reagents for diagnosing type 1 diabetes

Description:
RELATED APPLICATIONS 
     This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/879,347, filed Sep. 18, 2013; the contents of which are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Type 1 diabetes is caused by the destruction of pancreatic β cells due to an autoimmune process, which occurs over years. By the time clinical symptoms appear, the mass of β cells is reduced by at least 70-80% (Cnop, M. et al., (2005) Diabetes December; 54 Suppl. 2:S97-107). 
     A nested PCR diagnostic for diabetes has been developed that detects the relative amount of circulating un-methylated β cell insulin DNA as a result of cell death (Akirav, E. M. et. al., (2011) Proc. Natl. Acad. Sci. 108: 19018-19023). However, nested PCR produces biases and artifacts. 
     Accordingly new methods for diagnosing and monitoring type 1 diabetes are needed. 
     SUMMARY OF THE INVENTION 
     Featured herein are novel primers, probes and assays for diagnosing and monitoring type 1 diabetes. 
     The assays are highly sensitive and may be performed in a multiplexed fashion to diagnose diabetes before the onset of clinical symptoms and provide clinicians with a tool to decide for whom and when immune therapy might be useful. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURE 
         FIG. 1  depicts the cloning of L2_UM DNA and L2_M DNA into the pUC57 plasmid. The underlined sequences highlight the regions of primers and probes. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Described herein are specific primers and probes, which can be used to measure the relative amounts of methylated and un-methylated insulin DNA in serum obtained from a human subject. The un-methylated form of the gene expresses functional insulin, while the methylated form does not express protein. Since β cells are the only significant source of insulin gene expression, the assay is able to measure epigenetically circulating un-methylated insulin DNA as a marker for β cell death. Methylated insulin DNA is used for normalizing the varying levels of DNA between individual specimens. 
     
       
         
           
               
             
               
                 TABLE 1  
               
               
                   
               
               
                 List of primers and probes 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 L2_KH1_FWD4 
                 GTGGTTTATATTTGGTGGA (SEQ ID NO. 3) 
               
               
                   
               
               
                 L2_KH1_REV4 
                 ATTAACTCACCCTACAAATC (SEQ ID NO. 4) 
               
               
                   
               
            
           
         
       
     
     The nucleotide sequence of the unmethylated insulin DNA detection probe is: ATTTAAGATTTGTTGGGAGGTAGAG (SEQ ID NO: 1) and the nucleotide sequence of the methylated insulin DNA detection probe is: ATTTAAGATTCGTCGGGAGGTAGAG (SEQ ID NO: 2). One of skill in the art could alter the probes by deleting, replacing of altering one or more nucleotides without substantially changing probe function. 
     The probes target a region of the human insulin gene with 2 CpG sites (i.e. regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide). When DNA is treated with bisulfite, the un-methylated CpG is converted to TG (thymine, guanine), while a methylated CpG site is protected from this change. The two probes described herein target either a methylated or un-methylated insulin gene. 
     
       
         
           
               
            
               
                 Also described are double quencher probes specific 
               
               
                 for un-methylated human insulin gene DNA detection 
               
               
                 with fluorescein amidite (FAM): 
               
               
                 L2_KH1_um 
               
               
                 (SEQ ID NO: 3) 
               
               
                 5′-/56-FAM/ATTTAAGAT/ZEN/TTGTTGGGAGGTAGAG/ 
               
               
                   
               
               
                 3IABkFQ/-3′ 
               
               
                 and 
               
               
                   
               
               
                 for methylated human insulin gene detection 
               
               
                 with hexachloro-fluoroscein (HEX): 
               
               
                 L2_KH1_M 
               
               
                 (SEQ ID NO: 4) 
               
               
                 5′-/5HEX/ATTTAAGAT/ZEN/TCGTCGGGAGGTAGAG/ 
               
               
                   
               
               
                 3IABkFQ/-3′ 
               
            
           
         
       
     
     Use of these probes significantly reduces the background noise thereby increasing the noise to signal ratio. 
     The forward and reverse human insulin gene primers used in the examples are GTGGTTTATATTTGGTGGA (SEQ ID NO: 5) and ATTAACTCACCCTACAAATC (SEQ ID NO: 6). However, one of skill in the art may vary these sequences, e.g., by deleting, substituting or altering certain nucleotides without substantially impacting primer function. 
     The probes and primers described herein may be contacted with isolated and bisulfite-treated genomic DNA from an appropriate sample (e.g. serum, islet cells or peripheral blood mononuclear cells (PBMCs)). The reaction mixture may be loaded into a droplet generator. The droplets may be deposited on a plate and transferred into a polymerase chain reactor for amplification. The plate may then be transferred to a droplet reader for analysis of the data. 
     Use of the described probes and primers in a sensitive diagnostic assay, such as Droplet Digital Polymerase Chain Reaction (ddPCR), results in a highly specific and sensitive assay. In particular, as shown in the following examples, fewer than 13 copies were detected in a 25 ul PCR reaction with the target gene. In addition, in a recovery assay, where 5000 copies of L2_M plasmid representing bisulfite-treated methylated human insulin gene DNA were spiked with fewer than 10 copies of L2_UM plasmid representing bisulfite-treated un-methylated target DNA, L2_UM DNA was successfully detected. 
     As the results reported in the following example show, use of the described probes, primers and assays can be used to determine insulin DNA methylation status at low concentrations of DNA. 
     These primers and probes alone or in conjunction with instructions for use may be prepared as a kit for diagnosing or monitoring type 1 diabetes. 
     Table 1 lists additional probes and primers for amplification of methylation sensitive sites. Each assay has one set of probes, which can be used with 1 to 7 different sets of primers. Some probes will require modifications to the probes themselves to increase the annealing temperature and specificity such as—minor groove binder (MGB) modification, locked nucleic acid modification (LNA) and or Zen modification. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 List of Probes and Primers 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 5′-3′ 
                 Type (5′-3′) 
                 Sequence1 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 AGAGTTT TG TTTTGTA (SEQ ID NO: 7) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 AGAGTTTC G TTTTGTA (SEQ ID NO: 8) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGGATAGTAGTGTAA (SEQ ID NO: 9) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCTACTCACAACTAA (SEQ ID NO: 10) 
                 118 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence5 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 TGGTTATTGGGTTTT (SEQ ID NO: 11) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 TGGTTATCGGGTTTT (SEQ ID NO: 12) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGAAAGTGGTTTAGGTGAGGGTTT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 13) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CTCCTTAATCATCAACACCTCTTCCTC 
                 142 
               
               
                   
                 Primer 
                 (SEQ ID NO: 14) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TAGTTGTGAGTAGGGATAGGTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 15) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AATCTCCTTAATCATCAACACCT (SEQ ID 
                 97 
               
               
                   
                 Primer 
                 NO: 16) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGTTTAATGTGGAAAGTGGTTTAG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 17) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AACCATTTCCCTAATACTAAATCTATAA 
                 186 
               
               
                   
                 Primer 
                 (SEQ ID NO: 18) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGGTTTAATGTGGAAAGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 19) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCAAACCATTTCCCTAATAC (SEQ ID NO: 
                 192 
               
               
                   
                 Primer 
                 20) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GGGATAGTAGTGTAAAGAGTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 21) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 ACTACAATTTCCAAACCATTTC (SEQ ID 
                 245 
               
               
                   
                 Primer 
                 NO: 22) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence6 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 TTAATGATTTGTTGGTTTTGA (SEQ ID NO: 
                   
               
               
                   
                 Unmeth 
                 23) 
                   
               
               
                   
                 Probe_meth 
                 TTAATGATTCGTTGGTTTTGA (SEQ ID NO: 
                   
               
               
                   
                   
                 24) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGAAAGTGGTTTAGGTGAGGGTTT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 25) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ATCTCCTTAATCATCAACACCTCTTCC 
                 144 
               
               
                   
                 Primer 
                 (SEQ ID NO: 26) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GTGAGTAGGGATAGGTTTGGTTAT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 27) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCCTACCACCTAACCCATTAAA (SEQ ID 
                 197 
               
               
                   
                 Primer 
                 NO: 28) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGATAGGTTTGGTTATTGGGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 29) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 TCCAAACCATTTCCCTAATACTAAAT (SEQ 
                 119 
               
               
                   
                 Primer 
                 ID NO: 30) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TAGTTGTGAGTAGGGATAGGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 31) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACTACAATTTCCAAACCATTTC (SEQ ID 
                 143 
               
               
                   
                 Primer 
                 NO: 32) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTTAATGTGGAAAGTGGTTTAG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 33) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 AAATCTCCTTAATCATCAACAC (SEQ ID 
                 154 
               
               
                   
                 Primer 
                 NO: 34) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence7 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 AAGAGGTGTTGA TG ATTAAGGAGAT (SEQ ID 
                   
               
               
                   
                 Unmeth 
                 NO: 35) 
                   
               
               
                   
                 Probe_meth 
                 AAGAGGTGTTGAC G ATTAAGGAGAT (SEQ ID 
                   
               
               
                   
                   
                 NO: 36) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GTGAGTAGGGATAGGTTTGGTTAT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 37) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCCTACCACCTAACCCATTAAA (SEQ ID 
                 197 
               
               
                   
                 Primer 
                 NO: 38) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GAAAGTGGTTTAGGTGAGGGTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 39) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 TCCAAACCATTTCCCTAATACTAAATCT 
                 179 
               
               
                   
                 Primer 
                 (SEQ ID NO: 40) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TAGTTGTGAGTAGGGATAGGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 41) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACTACAATTTCCAAACCATTTC (SEQ ID 
                 143 
               
               
                   
                 Primer 
                 NO: 42) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GATAGGTTTGGTTATTGGGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 43) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 ATTTCCAAACCATTTCCCTAATA (SEQ ID 
                 121 
               
               
                   
                 Primer 
                 NO: 44) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTTAATGATTTGTTGGTTT (SEQ ID NO: 45) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCCACTAACTTTATAATCTC (SEQ ID NO: 
                 201 
               
               
                   
                 Primer 
                 46) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence8 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 AAATGGTT TG GAAATTGTA (SEQ ID NO: 47) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 AAATGGTT CG GAAATTGTA (SEQ ID NO: 48) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TGAGGAAGAGGTGTTGATGATT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 49) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCCTACCACCTAACCCATTAAA (SEQ ID 
                 136 
               
               
                   
                 Primer 
                 NO: 50) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 AGAGGTGTTGATGATTAAGGAGATT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 51) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACCTCTTCTAATACAACCTATCCTAAA 
                 223 
               
               
                   
                 Primer 
                 (SEQ ID NO: 52) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 AGTTGTGAGTAGGGATAGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 53) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCACTAACTTTATAATCTCAAA (SEQ ID 
                 247 
               
               
                   
                 Primer 
                 NO: 54) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGAGGAAGAGGTGTT (SEQ ID NO: 55) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 ACCTCTTCTAATACAACCTA (SEQ ID NO: 
                 231 
               
               
                   
                 Primer 
                 56) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTTATAGATTTAGTATTAGGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 57) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CTACTTAATAACCTCTTCT (SEQ ID NO: 58) 
                 206 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence9 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 TTAGG TG GTAGGG (SEQ ID NO: 59) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 TTAGG TG GTAGGG (SEQ ID NO: 60) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AATAACCTCTTCTAATACAACCTATCCT 
                 183 
               
               
                 Primer Pair 1 
                 Primer 
                 (SEQ ID NO: 61) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 AGTATTAGGGAAATGGTTTGGA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 62) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CAAACCTACTTAATAACCTCTTCTAAT 
                 200 
               
               
                   
                 Primer 
                 (SEQ ID NO: 63) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GAGGAAGAGGTGTTGATG (SEQ ID NO: 64) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCACTAACTTTATAATCTCAAA (SEQ ID 
                 181 
               
               
                   
                 Primer 
                 NO: 65) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTAGTATTAGGGAAATG (SEQ ID NO: 66) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 ACCTACTTAATAACCTC (SEQ ID NO: 67) 
                 200 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence10 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 AAGTTAG TG GGGGTTT (SEQ ID NO: 68) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 AAGTTAG CG GGGGTTT (SEQ ID NO: 69) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTAATGGGTTAGGTGGTAGGG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 70) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACCTCTTCTAATACAACCTATCCTAAA 
                 115 
               
               
                   
                 Primer 
                 (SEQ ID NO: 71) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGAAATGGTTTGGAAATTGTAGTT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 72) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACCTACTTAATAACCTCTTCTAATACAACC 
                 190 
               
               
                   
                 Primer 
                 (SEQ ID NO: 73) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GTATTAGGGAAATGGTTTGGAAAT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 74) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 ACAAACCTACTTAATAACCTCTTCTA (SEQ 
                 200 
               
               
                   
                 Primer 
                 ID NO: 75) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 AGTATTAGGGAAATGGT (SEQ ID NO: 76) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CAAACCTACTTAATAACC (SEQ ID NO: 77) 
                 200 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence12 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTTG TG TTAGGT  (SEQ ID NO: 78) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTTG CG TTAGGT  (SEQ ID NO: 79) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGTTGTATTAGAAGAGGTTATTAAGTAGGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 80) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CTTCACAAACCCAACCACATCC (SEQ ID 
                 131 
               
               
                   
                 Primer 
                 NO: 81) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTAGGATAGGTTGTATTAGAAGAGGTTATT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 82) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCAACCACATCCTCCCTACT (SEQ ID NO: 
                 129 
               
               
                   
                 Primer 
                 83) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TAATGGGTTAGGTGGTAGGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 84) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCACATACTTCACAAACC (SEQ ID NO: 
                 234 
               
               
                   
                 Primer 
                 85) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 AGGATAGGTTGTATTAGAAGA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 86) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAATCCAACCACCCTAAA (SEQ ID NO: 87) 
                 91 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTGAGATTATAAAGTTAGTGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 88) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 AAACCAAATACCCTACC (SEQ ID NO: 89) 
                 227 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence13 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TAGGGAGGA TG TGGTTGGGT  (SEQ ID NO: 
                   
               
               
                   
                 Unmeth 
                 90) 
                   
               
               
                   
                 Probe_meth 
                   TAGGGAGGA CG TGGTTGGGT  (SEQ ID NO: 
                   
               
               
                   
                   
                 91) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GTGTTAGGTGGGTTTAGGA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 92) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAACCAACAACACCAACAA (SEQ ID NO: 
                 223 
               
               
                   
                 Primer 
                 93) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 AGGATAGGTTGTATTAGAAGAGG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 94) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACCAAATACCCTACCTTAAA (SEQ ID 
                 182 
               
               
                   
                 Primer 
                 NO: 95) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTAGGGTGGTTGGATTT (SEQ ID NO: 96) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CACACAAATATTAATTCACAAA (SEQ ID 
                 250 
               
               
                   
                 Primer 
                 NO: 97) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence14 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGTTGG TG TTGTTGG  (SEQ ID NO: 98) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGTTGG CG TTGTTGG  (SEQ ID NO: 99) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGGATGTGGTTGGGTTTGTGA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 100) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AACTTCCACCAAATATAAACCACACAAATA 
                 231 
               
               
                   
                 Primer 
                 (SEQ ID NO: 101) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGTTGGGTTTGTGAAGTATGTG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 102) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACCAAATATAAACCACACAAATATTAATTC 
                 216 
               
               
                   
                 Primer 
                 (SEQ ID NO: 103) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTGTAGTAGGGAGGATGTGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 104) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACTTCCACCAAATATAAACCA (SEQ ID 
                 242 
               
               
                   
                 Primer 
                 NO: 105) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTAAGGTAGGGTATTTGGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 106) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCTCTACCTCCCAACAAATC (SEQ ID NO: 
                 248 
               
               
                   
                 Primer 
                 107) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GTGTTAGGTGGGTTTAGGA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 108) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 AAACTACAACTAAATCAAATCCC (SEQ ID 
                 250 
               
               
                   
                 Primer 
                 NO: 109) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 6 
                 Forward 
                 TTTAAGGTAGGGTATTTGGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 110) 
                   
               
               
                 Primer Pair 6 
                 Reverse 
                 ACCACACAAATATTAATTCACAAA (SEQ ID 
                 166 
               
               
                   
                 Primer 
                 NO: 111) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 7 
                 Forward 
                 TTGTAGTAGGGAGGATGTGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 112) 
                   
               
               
                 Primer Pair 7 
                 Reverse 
                 AAACTACAACTAAATCAAATCCC (SEQ ID 
                 200 
               
               
                   
                 Primer 
                 NO: 113) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                   
                 Sequence15 
                   
               
               
                   
               
               
                   
                 Probe- 
                   TTAGT TG TAGTT  (SEQ ID NO: 114) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTAGT CG TAGTT  (SEQ ID NO: 115) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGTTGGGTTTGTGAAGTATGTG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 116) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACCAAATATAAACCACACAAATATTAATTC 
                 216 
               
               
                   
                 Primer 
                 (SEQ ID NO: 117) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGTAGTAGGGAGGATGTGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 118) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTTCCACCAAATATAAACCA (SEQ ID 
                 242 
               
               
                   
                 Primer 
                 NO: 119) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTGTTGGTGTTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 120) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCTCTACCTCCCAACAAATC (SEQ ID NO: 
                 152 
               
               
                   
                 Primer 
                 121) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTAAGGTAGGGTATTTGGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 122) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 ACCACACAAATATTAATTCACAAA (SEQ ID 
                 166 
               
               
                   
                 Primer 
                 NO: 123) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence16 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGTG TG GTTTA  (SEQ ID NO: 124) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGTG CG GTTTA  (SEQ ID NO: 125) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTGTTGGTGTTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 126) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCTCTACCTCCCAACAAATC (SEQ ID NO: 
                 152 
               
               
                   
                 Primer 
                 127) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGATTTGATTTAGTTGTAGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 128) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CTCACCCTACAAATCCTCTAC (SEQ ID NO: 
                 142 
               
               
                   
                 Primer 
                 129) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTAAGGTAGGGTATTTGGTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 130) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 ACCTCCCAACAAATCTTAAATA (SEQ ID 
                 243 
               
               
                   
                 Primer 
                 NO: 131) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GTTGGGTTTGTGAAGTA (SEQ ID NO: 132) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCCACACACTAAATAAA (SEQ ID NO: 133) 
                 240 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence17 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGTGTG TG GGGAA  (SEQ ID NO: 134) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGTGTG CG GGGAA  (SEQ ID NO: 135) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 ATTTGTGTGGTTTATATTTGGTGGAAGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 136) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACTCACCCTACAAATCCTCTACCT (SEQ ID 
                 108 
               
               
                   
                 Primer 
                 NO: 137) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGTGAATTAATATTTGTGTGGTTTATATT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 138) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 TCCTCTACCTCCCAACAAATC (SEQ ID NO: 
                 106 
               
               
                   
                 Primer 
                 139) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTGTTGGTGTTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 140) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 TCTACCTCCCAACAAATCTTAAATA (SEQ 
                 150 
               
               
                   
                 Primer 
                 ID NO: 141) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGTGAATTAATATTTGTGTGGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 142) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CAATAAACAATTAACTCACCCTAC (SEQ ID 
                 134 
               
               
                   
                 Primer 
                 NO: 143) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GGGATTTGATTTAGTTGTAGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 144) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CTAAATAACAACCTCCTACCC (SEQ ID NO: 
                 240 
               
               
                   
                 Primer 
                 145) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence18 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGATT TG T TG GGAG  (SEQ ID NO: 146) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGATT CG T CG GGAG  (SEQ ID NO: 147) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 ATTTGTGTGGTTTATATTTGGTGGAAGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 148) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACAATTAACTCACCCTACAAATCCTCTAC 
                 115 
               
               
                   
                 Primer 
                 (SEQ ID NO: 149) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TGAATTAATATTTGTGTGGTTTATATTTGG 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 150) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACAATAAACAATTAACTCACCCTACA (SEQ 
                 131 
               
               
                   
                 Primer 
                 ID NO: 151) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTGTGAATTAATATTTGTGTGGTTTA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 152) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCTACTAAATAACAACCTCCTACC (SEQ ID 
                 222 
               
               
                   
                 Primer 
                 NO: 153) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TGTTGGTGTTGTTGGTTT (SEQ ID NO: 154) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCCTTCTACCCATACTAAATAAA (SEQ ID 
                 242 
               
               
                   
                 Primer 
                 NO: 155) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTTGTGAATTAATATTTGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 156) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCCTACTAAATAACAACC (SEQ ID NO: 
                 223 
               
               
                   
                 Primer 
                 157) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence20 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGGT TG TTTTT  (SEQ ID NO: 158) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGGT CG TTTTT  (SEQ ID NO: 159) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGTAGAGGATTTGTAGGGTGAGTTAAT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 160) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCCTACTAAATAACAACCTCCTACCC (SEQ 
                 125 
               
               
                   
                 Primer 
                 ID NO: 161) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGTTGGGAGGTAGAGGATTTGTA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 162) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACCAACACCATCCTCAAACTAAA (SEQ 
                 238 
               
               
                   
                 Primer 
                 ID NO: 163) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 AGGGTGAGTTAATTGTTTATTGTTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 164) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 ACATACCCACCCTCTAATATATCTCAA 
                 250 
               
               
                   
                 Primer 
                 (SEQ ID NO: 165) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TAAGATTTGTTGGGAGGTAGAG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 166) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCCTTCTACCCATACTAAATAAA (SEQ ID 
                 115 
               
               
                   
                 Primer 
                 NO: 167) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GTAGGGTGAGTTAATTGTTTATT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 168) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 AACCAACACCATCCTCA (SEQ ID NO: 169) 
                 216 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence21 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTTGG TG TTTTT  (SEQ ID NO: 170) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTTGG CG TTTTT  (SEQ ID NO: 171) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGTAGAGGATTTGTAGGGTGAGTTAAT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 172) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CCCTACTAAATAACAACCTCCTACCC (SEQ 
                 125 
               
               
                   
                 Primer 
                 ID NO: 173) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGTTGGGAGGTAGAGGATTTGTA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 174) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACCAACACCATCCTCAAACTAAA (SEQ 
                 238 
               
               
                   
                 Primer 
                 ID NO: 175) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 AGGGTGAGTTAATTGTTTATTGTTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 176) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 ACATACCCACCCTCTAATATATCTCAA 
                 250 
               
               
                   
                 Primer 
                 (SEQ ID NO: 177) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 AGATTTGTTGGGAGGTAGAG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 180) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACCAACACCATCCTCA (SEQ ID NO: 
                 242 
               
               
                   
                 Primer 
                 181) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GTAGGGTGAGTTAATTGTTTAT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 182) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCCTTCTACCCATACTAAAT (SEQ ID NO: 
                 88 
               
               
                   
                 Primer 
                 183) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence22 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGGTTA TG TTTTAA  (SEQ ID NO: 184) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGGTTA CG TTTTAA  (SEQ ID NO: 185) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGTAGGG 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 186) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAACCAACACCATCCTCAAACT (SEQ ID 
                 130 
               
               
                   
                 Primer 
                 NO: 187) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 AGGGTGAGTTAATTGTTTATTGTTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 188) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACATACCCACCCTCTAATATATCTCAA 
                 250 
               
               
                   
                 Primer 
                 (SEQ ID NO: 189) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGGTAGGAGGTTGTTATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 190) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 ACATACCCACCCTCTAATA (SEQ ID NO: 
                 165 
               
               
                   
                 Primer 
                 191) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTAGTATGGGTAGAAGGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 192) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAATTCTAACTAAACCACAAA (SEQ ID NO: 
                 124 
               
               
                   
                 Primer 
                 193) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence23 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGAGGA TG GTGTTG  (SEQ ID NO: 194) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGAGGA CG GTGTTG  (SEQ ID NO: 195) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 AGGGTGAGTTAATTGTTTATTGTTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 196) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACATACCCACCCTCTAATATATCTCAA 
                 250 
               
               
                   
                 Primer 
                 (SEQ ID NO: 197) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 198) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACATACCCACCCTCTAATA (SEQ ID NO: 
                 167 
               
               
                   
                 Primer 
                 199) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTGTGGTTTAGTTAGAATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 200) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCTCCTCCAAACATAA (SEQ ID NO: 201) 
                 250 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence24 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGGTTT TG GTAGTT  (SEQ ID NO: 201) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGGTTT CG GTAGTT  (SEQ ID NO: 201) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 AGGGTGAGTTAATTGTTTATTGTTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 202) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACATACCCACCCTCTAATATATCTCAA 
                 250 
               
               
                   
                 Primer 
                 (SEQ ID NO: 203) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 204) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACATACCCACCCTCTAATA (SEQ ID NO: 
                 167 
               
               
                   
                 Primer 
                 205) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTAGTTTGAGGATGGTGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 206) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCTCCTCCAAACATAATAAA (SEQ ID NO: 
                 229 
               
               
                   
                 Primer 
                 207) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGTGGTTTAGTTAGAATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 208) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACACTTAAATCTACAATCAT (SEQ ID 
                 161 
               
               
                   
                 Primer 
                 NO: 209) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence25 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGTTT TG AGATA  (SEQ ID NO: 210) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGTTT CG AGATA  (SEQ ID NO: 211) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTGAGGATGGTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 212) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCTCCTCCAAACATAATAA (SEQ ID 
                 227 
               
               
                   
                 Primer 
                 NO: 213) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 214) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACATACCCACCCTCTAATA (SEQ ID NO: 
                 167 
               
               
                   
                 Primer 
                 215) 
                   
               
               
                 Primer Pair 3 
                 Product 
                   
                   
               
               
                   
                 Forward 
                 TTTAGTTTGAGGATGGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 216) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACACTTAAATCTACAATCAT (SEQ ID 
                 142 
               
               
                   
                 Primer 
                 NO: 217) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TGTGGTTTAGTTAGAATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 218) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AATCTACAATCATCAAATAA (SEQ ID NO: 
                 150 
               
               
                   
                 Primer 
                 219) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence26 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGGGTA TG TTTTT  (SEQ ID NO: 220) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGGGTA CG TTTTT  (SEQ ID NO: 221) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTGAGGATGGTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 222) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCTCCTCCAAACATAATAA (SEQ ID 
                 227 
               
               
                   
                 Primer 
                 NO: 223) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGTAGG (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 224) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACACTTAAATCTACAATCATCAAATAAA 
                 247 
               
               
                   
                 Primer 
                 (SEQ ID NO: 225) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGGTAGGAGGTTGTTATT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 226) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACACTTAAATCTACAATCAT (SEQ ID 
                 247 
               
               
                   
                 Primer 
                 NO: 227) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTAGTTTGAGGATGGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 228) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CAAACAAACAACCACAC (SEQ ID NO: 229) 
                 247 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence27 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTATT TG TTTTT  (SEQ ID NO: 230) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTATT CG TTTTT  (SEQ ID NO: 231) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTGAGATATATTAGAGGGTGGGTATGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 232) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AACCCACTCAAACAAACAACCA (SEQ ID 
                 223 
               
               
                   
                 Primer 
                 NO: 233) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGAGGATGGTGTTGGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 234) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CACCCTCCTCCAAACATAATAA (SEQ ID 
                 227 
               
               
                   
                 Primer 
                 NO: 235) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGGTAGGAGGTTGTTATTTAGTAGG (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 236) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACACTTAAATCTACAATCATCAAATAAA 
                 247 
               
               
                   
                 Primer 
                 (SEQ ID NO: 237) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GGGTAGGAGGTTGTTATT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 238) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACACTTAAATCTACAATCAT (SEQ ID 
                 247 
               
               
                   
                 Primer 
                 NO: 239) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence28 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGTTT TG TAGTT  (SEQ ID NO: 240) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGTTT CG TAGTT  (SEQ ID NO: 241) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATATATTAGAGGGTGGGTATGTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 242) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACACCCTCCTCCAAACATAATAAA (SEQ ID 
                 199 
               
               
                   
                 Primer 
                 NO: 243) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGATAGTAGTGTAAAGAGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 244) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCTATCCCTACTCACAACTAAA (SEQ ID 
                 124 
               
               
                   
                 Primer 
                 NO: 245) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TGAGGATGGTGTTGGTT (SEQ ID NO: 246) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CACTTAAATCTACAATCATCAAATAAA 
                 132 
               
               
                   
                 Primer 
                 (SEQ ID NO: 247) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGTGGTTTAGTTAGAATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 24\8) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACACTTAAATCTACAATCAT (SEQ ID 
                 161 
               
               
                   
                 Primer 
                 NO: 249) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence29 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   ATGAT TG TAGA  (SEQ ID NO: 250) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   ATGAT CG TAGA  (SEQ ID NO: 251) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATATATTAGAGGGTGGGTATGTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 252) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACACCCTCCTCCAAACATAATAAA (SEQ ID 
                 199 
               
               
                   
                 Primer 
                 NO: 253) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence30 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGTTTTA TG TTGTTTGT  (SEQ ID NO: 254) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGTTTTA CG TTGTTTGT  (SEQ ID NO: 255) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATATATTAGAGGGTGGGTATGTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 256) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACACCCTCCTCCAAACATAATAAA (SEQ ID 
                 199 
               
               
                   
                 Primer 
                 NO: 257) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTATTTGATGATTGTAGATTTAAGTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 258) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACAACCACACCCTCCT (SEQ ID NO: 
                 130 
               
               
                   
                 Primer 
                 259) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TATTTGATGATTGTAGATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 260) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCATCTCCTAACTATAA (SEQ ID NO: 261) 
                 189 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence31 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGGG TG AATAT  (SEQ ID NO: 262) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGGG CG AATAT  (SEQ ID NO: 263) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATATATTAGAGGGTGGGTATGTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 264) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACACCCTCCTCCAAACATAATAAA (SEQ ID 
                 199 
               
               
                   
                 Primer 
                 NO: 265) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTATTTGATGATTGTAGATTTAAGTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 266) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACAACCACACCCTCCT (SEQ ID NO: 
                 130 
               
               
                   
                 Primer 
                 267) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TATTTGATGATTGTAGATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 268) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCATCTCCTAACTATAA (SEQ ID NO: 269) 
                 189 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence32 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   ATTA TG TT TG GAGG  (SEQ ID NO: 270) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   ATTA CG TT CG GAGG  (SEQ ID NO: 271) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTATTTGATGATTGTAGATTTAAGTGTTT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 272) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CAAACAAACAACCACACCCT (SEQ ID NO: 
                 135 
               
               
                   
                 Primer 
                 273) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TATTTGATGATTGTAGATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 274) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCCATCTCCTAACTATAA (SEQ ID NO: 275) 
                 189 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence33 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGGAGGG TG TGGTTG  (SEQ ID NO: 276) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGGAGGG CG TGGTTG  (SEQ ID NO: 277) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTGATGATTGTAGATTTAAGTGTTT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 278) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAATCTAACCCACTCAAACAAA (SEQ ID 
                 144 
               
               
                   
                 Primer 
                 NO: 279) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TATTTGATGATTGTAGATTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 280) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCCATCTCCTAACTATAA (SEQ ID NO: 281) 
                 189 
               
               
                   
                 Primer 
                   
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence34 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTTTGT TG TTAGG  (SEQ ID NO: 282) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTTTGT CG TTAGG  (SEQ ID NO: 283) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TGGTTGTTTGTTTGAGTGGGTTAGA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 284) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACCCACATATCCCACCTCCTT (SEQ ID NO: 
                 174 
               
               
                   
                 Primer 
                 285) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTGGAGGAGGGTGTGGTTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 286) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AACCTAAACAAATAATCCCAATACTTCTCC 
                 138 
               
               
                   
                 Primer 
                 (SEQ ID NO: 287) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GAGGGTGTGGTTGTTTGTTTGA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 288) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCAACACCCACATATCCCA (SEQ ID NO: 
                 187 
               
               
                   
                 Primer 
                 289) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTATGTTTGGAGGAGGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 290) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACCTAAACAAATAATCCCAATA (SEQ ID 
                 144 
               
               
                   
                 Primer 
                 NO: 291) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TGATTGTAGATTTAAGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 292) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCCATCTCCTAACTATAAA (SEQ ID NO: 
                 182 
               
               
                   
                 Primer 
                 293) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence35 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTTTA TG GTAG  (SEQ ID NO: 294) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTTTA CG GTAG  (SEQ ID NO: 295) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TGGTTGTTTGTTTGAGTGGGTTAGA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 296) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACCCACATATCCCACCTCCTT (SEQ ID NO: 
                 174 
               
               
                   
                 Primer 
                 297) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTGGAGGAGGGTGTGGTTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 298) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AACCTAAACAAATAATCCCAATACTTCTCC 
                 138 
               
               
                   
                 Primer 
                 (SEQ ID NO: 299) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GAGGGTGTGGTTGTTTGTTTGA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 300) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCCAACACCCACATATCCCA (SEQ ID NO: 
                 187 
               
               
                   
                 Primer 
                 301) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTATGTTTGGAGGAGGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 302) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 AAACCTAAACAAATAATCCCAATA (SEQ ID 
                 144 
               
               
                   
                 Primer 
                 NO: 303) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TGATTGTAGATTTAAGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 304) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCCATCTCCTAACTATAAA (SEQ ID NO: 
                 182 
               
               
                   
                 Primer 
                 305) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence36 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGTGGG TG TTGGG  (SEQ ID NO: 306) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGTGGG CG TTGGG  (SEQ ID NO: 307) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGGAGAAGTATTGGGATTATTTGTTTAGGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 308) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACACTCCCACCCATCTCCAA (SEQ ID NO: 
                 164 
               
               
                   
                 Primer 
                 309) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGAAGGAGGTGGGATA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 310) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 CCATCTCCAACCAAACTAAAC (SEQ ID NO: 
                 97 
               
               
                   
                 Primer 
                 311) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GGGAGAAGTATTGGGATTATTTG (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 312) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CTACCCACCAACCCTAAATC (SEQ ID NO: 
                 184 
               
               
                   
                 Primer 
                 313) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 TTTGTTTGAGTGGGTTAGA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 314) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CCACACTAAATATAAACCTACAA (SEQ ID 
                 199 
               
               
                   
                 Primer 
                 NO: 315) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 TTTATAGTTAGGAGATGGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 316) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 CCAAACTAAACCCAAATTA (SEQ ID NO: 
                 186 
               
               
                   
                 Primer 
                 317) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence37 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTAGTT TG GTTGG  (SEQ ID NO: 318) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTAGTT CG GTTGG  (SEQ ID NO: 319) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGAGGTGGGATATGTGGGTGT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 320) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACCTACCCACCAACCCTAAATCA (SEQ ID 
                 124 
               
               
                   
                 Primer 
                 NO: 321) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGAGAAGTATTGGGATTATTTGTTTAGGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 322) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 ACAATACCCACCTACCCACCAA (SEQ ID 
                 195 
               
               
                   
                 Primer 
                 NO: 323) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GTTTGTAGGTTTATATTTAGTGTGGGTGAT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 324) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CTAAATCACACTCCCACCCATCT (SEQ ID 
                 84 
               
               
                   
                 Primer 
                 NO: 325) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GGGAAGGAGGTGGGATA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 326) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 ACCCTAAATCACACTCCCA (SEQ ID NO: 
                 117 
               
               
                   
                 Primer 
                 327) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 5 
                 Forward 
                 GGGAGAAGTATTGGGATTA (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 328) 
                   
               
               
                 Primer Pair 5 
                 Reverse 
                 AAACACAATACCCACCTA (SEQ ID NO: 
                 199 
               
               
                   
                 Primer 
                 329) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence38 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGTG TG ATTTA  (SEQ ID NO: 330) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGTG CG ATTTA  (SEQ ID NO: 331) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GGGAGAAGTATTGGGATTATTTGTTTAGGT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 332) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 ACAATACCCACCTACCCACCAA (SEQ ID 
                 195 
               
               
                   
                 Primer 
                 NO: 333) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 AATTTGGGTTTAGTTTGGTTGGAGA (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 334) 
                   
               
               
                 Primer Pair 2 
                 Probe 
                 AGTGTGATTTA (SEQ ID NO: 335) 
                   
               
               
                   
                 Reverse 
                 CACCCAACTCCACCTACCC (SEQ ID NO: 
                 169 
               
               
                 Primer Pair 3 
                 Primer 
                 336) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTAATTTGGGTTTAGTTTGG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 337) 
                   
               
               
                 Primer Pair 4 
                 Probe 
                 AGTGTGATTTA (SEQ ID NO: 338) 
                   
               
               
                   
                 Reverse 
                 AACACAATACCCACCTAC (SEQ ID NO: 
                 75 
               
               
                 Primer Pair 4 
                 Primer 
                 339) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence39 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGG CG GGTAG  (SEQ ID NO: 340) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGG CG GGTAG  (SEQ ID NO: 341) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATGGGTGGGAGTGTGATTTA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 342) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCAACTCCACCTACCC (SEQ ID NO: 
                 148 
               
               
                   
                 Primer 
                 343) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGGAGATGGGTGGGAGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 344) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA(SEQ ID 
                 180 
               
               
                   
                 Primer 
                 NO: 345) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence40 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   AGG TG GGTA  (SEQ ID NO: 346) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   AGG CG GGTA  (SEQ ID NO: 347) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATGGGTGGGAGTGTGATTTA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 348) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCAACTCCACCTACCC (SEQ ID NO: 
                 148 
               
               
                   
                 Primer 
                 349) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGGAGATGGGTGGGAGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 350) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA (SEQ ID 
                 180 
               
               
                   
                 Primer 
                 NO: 351) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GATTTAGGGTTGGTGGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 352) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CCACAATACCACACTTCTACA (SEQ ID NO: 
                 200 
               
               
                   
                 Primer 
                 353) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence41 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGTTT TG T TG TTGTT  (SEQ ID NO: 354) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGTTT CG T CG TTGTT  (SEQ ID NO: 355) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATGGGTGGGAGTGTGATTTA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 356) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCAACTCCACCTACCC(SEQ ID NO: 
                 148 
               
               
                   
                 Primer 
                 357) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTAGGGTTGGTGGGTAGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 358) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 TTCCACAATACCACACTTCTACAAA (SEQ 
                 200 
               
               
                   
                 Primer 
                 ID NO: 359) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTGGAGATGGGTGGGAGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 360) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA (SEQ ID 
                 180 
               
               
                   
                 Primer 
                 NO: 361) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GTAGGTGGGTATTGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 362) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CTAATACAACATTATTCCACAAT (SEQ ID 
                 200 
               
               
                   
                 Primer 
                 NO: 363) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence42 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TGTTT TG GAAT  (SEQ ID NO: 364) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TGTTT CG GAAT  (SEQ ID NO: 365) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATGGGTGGGAGTGTGATTTA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 366) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCAACTCCACCTACCC (SEQ ID NO: 
                 148 
               
               
                   
                 Primer 
                 367) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTAGGGTTGGTGGGTAGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 368) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 TTCCACAATACCACACTTCTACAAA (SEQ 
                 200 
               
               
                   
                 Primer 
                 ID NO: 369) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTGGAGATGGGTGGGAGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 370) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA (SEQ ID 
                 180 
               
               
                   
                 Primer 
                 NO: 371) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GTAGGTGGGTATTGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 372) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CTAATACAACATTATTCCACAAT (SEQ ID 
                 200 
               
               
                   
                 Primer 
                 NO: 373) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence43 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTTTG TGTG GTATGTTTT  (SEQ ID NO: 374) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTTTG CGCG GTATGTTTT  (SEQ ID NO: 375) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GAGATGGGTGGGAGTGTGATTTA (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 376) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 CACCCAACTCCACCTACCC (SEQ ID NO: 
                 148 
               
               
                   
                 Primer 
                 377) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTAGGGTTGGTGGGTAGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 378) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 TTCCACAATACCACACTTCTACAAA (SEQ 
                 200 
               
               
                   
                 Primer 
                 ID NO: 379) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTGGAGATGGGTGGGAGTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 380) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA (SEQ ID 
                 180 
               
               
                   
                 Primer 
                 NO: 381) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 4 
                 Forward 
                 GTAGGTGGGTATTGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 382) 
                   
               
               
                 Primer Pair 4 
                 Reverse 
                 CTAATACAACATTATTCCACAAT (SEQ ID 
                 200 
               
               
                   
                 Primer 
                 NO: 383) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence45 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TTGGG TG GGGGT  (SEQ ID NO: 384) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TTGGG CG GGGGT  (SEQ ID NO: 385) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTAGGGTTGGTGGGTAGGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 386) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 TTCCACAATACCACACTTCTACAAA (SEQ 
                 200 
               
               
                   
                 Primer 
                 ID NO: 387) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GATGGGTGGGAGTGTGATT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 388) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTACAAACTACCTACACCAAA (SEQ ID 
                 175 
               
               
                   
                 Primer 
                 NO: 389) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 GTAGGTGGGTATTGTGTTT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 390) 
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CTAATACAACATTATTCCACAAT (SEQ ID 
                 200 
               
               
                   
                 Primer 
                 NO: 391) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence46 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                 TAGAAGTGTGGTA (SEQ ID NO: 392) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                 TAGAAGCGTGGTA (SEQ ID NO: 393) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TTTGGTGTAGGTAGTTTGTAGTTT (SEQ ID 
                   
               
               
                   
                 Primer 
                 NO: 394) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AACTTTATTCCATCTCTCTCAATACA (SEQ 
                 182 
               
               
                   
                 Primer 
                 ID NO: 395) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 GGGTAGGTGGAGTTGGGT(SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 396) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AACAAATACTAATACAACATTATTCCACAA 
                 112 
               
               
                   
                 Primer 
                 (SEQ ID NO: 397) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 3 
                 Forward 
                 TTTGGTGTAGGTAGTTT (SEQ ID NO: 398) 
                   
               
               
                   
                 Primer 
                   
                   
               
               
                 Primer Pair 3 
                 Reverse 
                 CAATAATTCTCCAACTAATAAA (SEQ ID 
                 113 
               
               
                   
                 Primer 
                 NO: 399) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence47 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   ATTAGA TG TAGTT  (SEQ ID NO: 400) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   ATTAGA CG TAGTT  (SEQ ID NO: 401) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TGTAGAAGTGTGGTATTGTGGAATAAT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 402) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAACTTTATTCCATCTCTCTCAATACAAA 
                 140 
               
               
                   
                 Primer 
                 (SEQ ID NO: 403) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTGTAGAAGTGTGGTATTGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 404) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTTTATTCCATCTCTCTCA (SEQ ID 
                 142 
               
               
                   
                 Primer 
                 NO: 405) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence48 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   TAGTT TG TAGG  (SEQ ID NO: 406) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   TAGTT CG TAGG  (SEQ ID NO: 407) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 TGTAGAAGTGTGGTATTGTGGAATAAT 
                   
               
               
                   
                 Primer 
                 (SEQ ID NO: 408) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAACTTTATTCCATCTCTCTCAATACAAA 
                 140 
               
               
                   
                 Primer 
                 (SEQ ID NO: 409) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTTGTAGAAGTGTGGTATTGT (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 410) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTTTATTCCATCTCTCTCA (SEQ ID 
                 142 
               
               
                   
                 Primer 
                 NO: 411) 
                   
               
               
                   
                 Product 
               
               
                   
               
               
                   
                 Type 
                 Sequence49 
                 Amplicon 
               
               
                   
               
               
                   
                 Probe- 
                   ATT TG T TG TTTT  (SEQ ID NO: 412) 
                   
               
               
                   
                 Unmeth 
                   
                   
               
               
                   
                 Probe_meth 
                   ATT CG T CG TTTT  (SEQ ID NO: 413) 
                   
               
               
                 Primer Pair 1 
                 Forward 
                 GTAGAAGTGTGGTATTGTGGAATAAT (SEQ 
                   
               
               
                   
                 Primer 
                 ID NO: 414) 
                   
               
               
                 Primer Pair 1 
                 Reverse 
                 AAACTTTATTCCATCTCTCTCAATACA 
                 139 
               
               
                   
                 Primer 
                 (SEQ ID NO: 415) 
                   
               
               
                   
                 Product 
                   
                   
               
               
                 Primer Pair 2 
                 Forward 
                 TTGTAGAAGTGTGGTATTG (SEQ ID NO: 
                   
               
               
                   
                 Primer 
                 416) 
                   
               
               
                 Primer Pair 2 
                 Reverse 
                 AAACTTTATTCCATCTCTCT (SEQ ID NO: 
                 141 
               
               
                   
                 Primer 
                 417) 
                   
               
               
                   
                 Product 
               
               
                   
               
            
           
         
       
     
     Exemplification 
     The invention now being generally described, will be more readily understood by reference to the following example, which is included merely for purposes of illustration and is not intended to be limiting. 
     Experimental Design 
     All CpG sites for the human insulin gene (hg19_knownGene_uc021qcd.1 range=chr11:2181009-2182439) were mapped. The sequence was then transformed to a sequence representing bisulfite-treated DNA, where all Cs are converted to Ts and in the case of a methylated CpG site, the C is protected from conversion to T. The probes were designed to include two CpG sites at nucleotides 21814010 and 21814012 (http://genome.ucsc.edu/cgi-bin/hgGateway, Feb 2009 GRCh37/hgl9) in positions +396 and +399 from the transcription start site. 
     
       
         
           
               
            
               
                 The nucloetide sequence of the methylation sensi- 
               
               
                 tive probe is: 
               
               
                 (SEQ ID NO: 1) 
               
               
                 ATTTAAGATTTGTTGGGAGGTAGAG 
               
               
                 and 
               
               
                   
               
               
                 the nucleotide sequence of the methylation 
               
               
                 insensitive probe is: 
               
               
                 (SEQ ID NO: 2) 
               
               
                 ATTTAAGATTCGTCGGGAGGTAGAG. 
               
               
                   
               
               
                 The sequences of the forward and reverse primers 
               
               
                 are: 
               
               
                 (SEQ ID NO: 5) 
               
               
                 GTGGTTTATATTTGGTGGA 
               
               
                 and 
               
               
                   
               
               
                 (SEQ ID NO: 6) 
               
               
                 ATTAACTCACCCTACAAATC. 
               
            
           
         
       
     
     To increase sensitivity, specificity and to reduce background fluorescence, the probes were designed with an internal Zen quencher (ZEN) in addition to the 3′ Iowa Black FQ quencher (3IABkFQ). The ZEN probes were synthesized by Integrated DNA Technologies. 
     The synthetic L2_M and L2_UM sequences, which are replicas of bisulfite-treated methylated and un-methylated human insulin gene sequences (region 2181155-2181465 on Chr11), respectively, were cloned into pUC57 plasmids (Genewiz, Inc). 
       FIG. 1  illustrates the cloned sequences in pUC57 Kan plasmid. The plasmids were used for optimization of PCR conditions and to determine the sensitivity and specificity of the primers and probes. 
     Reaction mixtures of 25 ul volume, comprising 1× Droplet PCR supermix, (BioRad), 900 uM primer mix, 250 uM probe mix, 5 ul of plasmid (copy number ranging from 100,000 to 1 and mixed populations of un-methylated and methylated plasmids) or 4 ul of bisulfite treated genomic DNA from either serum, islet cells or peripheral blood mononuclear cells (PBMCs) was prepared. Twenty μl of the PCR reaction mix was loaded into a separate well of a eight channel droplet generator cartridge, and in a separate corresponding well 70 ul of droplet generation oil (BioRad) was loaded. The cartridge was loaded into a droplet generator (Biorad). Forty ul of the generated droplets were carefully transferred to a 96 well PCR plate and the PCR reaction was run on a thermal cycle with the following protocol: 10 min activation at 95° C. followed by 40 cycles of a two-step amplification protocol of 30 seconds at 94° C. denaturation and 60 seconds at 58° C. for a combined annealing-extension step. A final 10 min at 98° C. inactivation step completed the reaction. 
     The PCR plate was then transferred to a QX100 droplet reader (Biorad), which automatically reads the droplets from each well of the plate. Analysis of ddPCR data was performed using QuantaSoft Analysis software. 
     Islet, PBMCs and serum samples from non-diabetic and diabetic patients were used to validate the protocol developed using the plasmids. DNA was isolated using the Qiagen blood and tissue DNA extraction kit. DNA was bisulfite treated using an EZ DNA Methylation kit (Zymo Research, Irvine, Calif.). 
     Limit of detection assays were performed with plasmids, islets and PBMCs. The plasmid suspensions were made in a series of 10 fold serial dilutions from 100,000 copies to 1 copy. Bisulfite-treated DNA from islets and PBMCs were diluted over a ¼dilution series up to 1/1024 with amounts ranging from 148 ng to 0.1 ng. 
     Results 
     In the multiplexed assay, the probe for unmethylated human insulin gene DNA successfully detected 1 copy/μl of plasmid and showed no cross amplification with the plasmid representing methylated DNA. The probe for methylated DNA successfully detected 1 copy/ul per reaction and showed minimal cross amplification only with high numbers of plasmid representing un-methylated insulin gene DNA. 
     In the recovery assay, where approximately 3000 copies/ul of L2_M plasmid were mixed with 10 copies of the L2_UM plasmid, the ddPCR successfully detected the L2_UM plasmid successfully. 
     Using human islet tissue and PBMCs, we were able to detect unmethylated insulin DNA in as low as 0.1 ng of DNA. Also, the ratio of methylated insulin DNA to unmethylated insulin DNA did not change over varying concentrations. We then tested the assay with clinical samples, and were able to detect unmethylated DNA in pre-diabetics. Also, the ratio of unmethylated insulin coding DNA to methylated insulin DNA was significantly elevated in pre-diabetic (mean 0.4264±0.04034 N=6) patients compared to non-diabetic human controls (0.2122±0.02449 N=13). 
     Equivalents 
     While specific embodiments have been discussed, the above specification is intended to be illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification.