# Patent

## Title
Method for multiplexed nucleic acid patch polymerase chain reaction

## Abstract

The invention encompasses a method for amplifying at least two different nucleic acid sequences. In particular, the method encompasses a multiplexed nucleic acid patch polymerase chain reaction.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Ser. No. 13/556,590, filed Jul. 24, 2012, now U.S. Pat. No. 8,936,912, which is a continuation of U.S. application Ser. No. 12/555,627, filed Sep. 8, 2009, now U.S. Pat. No. 8,586,310, which claims the priority of U.S. provisional application No. 61/094,660, filed Sep. 5, 2008, each of which is hereby incorporated by reference in its entirety.


GOVERNMENTAL RIGHTS
This invention was made with government support under 5P50HG003170-0 awarded by the National Institutes of Health. The government has certain rights in the invention.


FIELD OF THE INVENTION
The invention encompasses a method for a amplifying at least two different nucleic acid sequences.
REFERENCE TO SEQUENCE LISTING
A paper copy of the sequence listing and a computer readable form of the same sequence listing are appended below and herein incorporated by reference. The information recorded in computer readable form is identical to the written sequence listing, according to 37 C.F.R. 1.821 (f).
BACKGROUND OF THE INVENTION
As the genes involved in various aspects of human physiology are elucidated, there are increasingly more candidate genes associated with disease. The application of this knowledge both in the clinic and to clinical research can be very powerful as the field moves toward personalized medicine. Examples of success include the sequencing of candidate disease loci in targeted populations, such as Ashkenazi Jews (Weinstein 2007), the sequencing of variants in drug metabolism genes to adjust dosage (Marsh and McLeod 2006), and the identification of genetic defects in cancer that make tumors more responsive to certain treatments (Marsh and McLeod 2006). However, the sequencing of many candidate genes across many individual samples necessitates the development of new technology to lower the cost and increase the throughput of medical re-sequencing to make clinical application more feasible.
The cost of sequencing is declining rapidly due to second generation sequencing technologies that perform a large number of sequencing reactions in parallel while using a small amount of reagent per reaction (Metzker 2005). These technologies integrate cloning and amplification into the sequencing protocol, which is essential for achieving the greater than 100-fold cost savings over traditional methods. However, this integration results in a loss of flexibility—it is not yet feasible to sequence a subset of the human genome in a large number of samples for the same cost as sequencing the complete genome of a single individual. This is a limitation, because sequencing the complete genome of a large numbers of individuals is still cost prohibitive, and the whole genome sequence of only a few individuals does not provide enough statistical power to make correlations between genotype and phenotype. The promise of personalized medicine based on genome analysis still glows on the horizon, but the significance behind observed variability is dim without an affordable technology to drive the necessary depth of patient sampling.
Current methods for analyzing sequence variation in a subset of the human genome rely on PCR to amplify the targeted sequences (Greenman et al. 2007; Sjoblom et al. 2006; Wood et al. 2007). Efforts to multiplex PCR have been hampered by the dramatic increase in the amplification of mispriming events as more primer pairs are used (Fan et al. 2006). In addition, large numbers of primer pairs often result in inter-primer interactions that prevent amplification (Han et al. 2006). Therefore, separate PCRs for each region of interest are performed, a costly approach when hundreds of individual PCRs must be performed for each sample (Greenman et al. 2007; Sjoblom et al. 2006; Wood et al. 2007). Furthermore, this strategy requires a large amount of starting DNA to supply enough template for all of the individual PCR reactions. This can be a problem as DNA is often a limiting factor when working with clinical samples.
It is important to choose the appropriate strategy for sample tracking to fully harness the throughput of second generation sequencing technologies. The sequencing capacities of these platforms are large enough that multiple samples can be sequenced with a single instrument run. To do this, one can use a separate compartment for each sample, but this only allows for a small number of samples, and there is a reduction in the total amount of sequence generated per run. Recently, Parameswaran et al. (Parameswaran et al. 2007) demonstrated the power of using DNA barcodes to label samples so that they can be pooled and sequenced together on the 454/Roche GS20 Sequencer. They were able to utilize the full capacity of the instrument and still determine from which sample each read originated. To realize the full power of second generation sequencing technologies, a multiplexing strategy should be compatible with DNA barcoding to track samples.
Therefore, there remains a need in the art for a multiplexed PCR method that simultaneously amplifies many targeted regions from a small amount of nucleic acid. The PCR method should also be compatible with next generation high throughput sequencing technologies where numerous samples can be processed in a single run. The PCR method should be specific and sensitive enough for identifying SNPs and mutations in individual samples.
SUMMARY OF THE INVENTION
One aspect of the present invention encompasses a method of amplifying at least two different nucleic acid sequences. Generally speaking, the method comprises the following steps: (a) defining the ends of at least two nucleic acid sequences; (b) annealing upstream and downstream nucleic acid patches to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch; (c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and (d) amplifying the nucleic acid sequences of step (c).
Another aspect of the invention encompasses a method of detecting short tandem repeats. The method typically comprises the following steps: (a) defining the ends of at least two nucleic acid sequences, wherein the ends are immediately adjacent to a short tandem repeat; (b) annealing upstream and downstream nucleic acid patches to each nucleic acid sequence of step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch; (c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; (d) amplifying the nucleic acid sequences of step (c); and (e) sequencing the products of step (d) to detect the length of the short tandem repeat.
Other aspects and iterations of the invention are described more thoroughly below.



BRIEF DESCRIPTION OF THE FIGURES
The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.
FIG. 1A-D depicts a schematic of nucleic acid patch PCR. (FIG. 1A) A PCR reaction containing primers pairs for all targets is performed on genomic DNA. The primers contain uracil substituted for thymine. The primers are then cleaved from the amplicons by the addition of heat-labile Uracil DNA Glycosylase, Endonuclease VIII, and single strand specific Exonuclease I. (FIG. 1B) The ends of the target regions are now internal to the PCR primers (nested). (FIG. 1C) Nucleic acid patch oligonucleotides are annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. (FIG. 1D) The selected amplicons are then amplified together simultaneously by PCR with universal primers.
FIG. 2 depicts a schematic of Sample Specific Barcode PCR. Sample-specific DNA barcodes are incorporated into the primers that are used for the final universal PCR. The 5′ end of the universal primer (white) is tailed with the sequences for the Roche/454 FLX Machine (grey) and sample-specific DNA sequences (black). When sequencing from either 454A or 454B, the first few bases indicate from which sample the read originated.
FIG. 3A-C shows the quantification of the abundance and reproducibility of nucleic acid patch PCR per exon in each sample. (FIG. 3A) Uniform Exon Abundance. Graph of the number of reads obtained for each targeted exon from the colon cancer sample and adjacent normal tissue. The 90 exons for which at least 1 read was obtained are ordered by abundance in the normal sample on the x-axis. The median number of reads per exon is 145. Seventy-six percent of all exons fell within 5 fold coverage of this median. All exons are within 3 log 10 of each other. (FIG. 3B) Correlation of number of reads across samples. Each exon is depicted as a point on the graph, where the x-axis is the number of reads in the normal sample and the y-axis is the number of reads in the colon cancer sample. The correlation was high (R2 of 93%), indicating high reproducibility across samples. (FIG. 3C) Fold difference in abundance across samples. We computed the fold change of abundance per exon between the two samples. 85% (77/90) of exons displayed a 2 fold or less difference in abundance between samples. 100% of exons displayed a 3 fold or less difference in abundance between samples. Dotted line indicates 3 fold change.
FIG. 4 depicts a schematic of bisulfite nucleic acid patch PCR with ends defined by AluI digest. Genomic DNA is digested with AluI restriction enzyme. Nucleic acid patch oligonucleotides are then annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. The reactions are then treated with sodium bisulfite to convert unmethylated cytosines to uracil. The selected amplicons are then amplified together simultaneously by PCR with universal primers.
FIG. 5 shows an image of the agarose gel electrophoresis of the final Universal PCR products of bisulfite nucleic acid patch PCR with ends defined by AluI digest. Each reaction was performed using decreasing quantities of starting human genomic DNA, as labeled in the figure. The expected smear of products is seen in the lanes that contained 900, 675, 450, 225, 112, 70, 50, and 20 ng of genomic DNA. The first lane contains Low Molecular Weight Ladder (NEB), with band sizes denoted on the left.
FIG. 6 depicts the sequencing results of bisulfite nucleic acid patch PCR with ends defined by AluI digest. The Y axis on the graph represents the number of reads obtained for each promoter. The promoters are order by length (bp) on the X axis.
FIG. 7A-G depicts a schematic of multiplexed bisulfite PCR. (FIG. 7A) Genomic DNA. (FIG. 76) Restriction digest. (FIG. 7C) Anneal patch oligos and universal primers specifically to the ends of desired fragments. (FIG. 7D) Ligate universal primers (U1 & U2) to targeted fragments. (FIG. 7E) Degrade unselected DNA with exonucleases. Targeted loci are protected from exonuclease by 3-prime modification on U2. (FIG. 7F) Treat with sodium bisulfite to convert unmethylated cytosine to uracil, leaving methylated cytosine intact. (FIG. 7G) PCR all loci simultaneously with universal primers tailed with sample-specific-DNA barcodes and sequencing machine primers (454A & 454B). Pool PCR products from all samples together for sequencing.
FIG. 8 depicts a photograph of an agarose gel showing that multiplexed bisulfite PCR works from small quantities of human genomic DNA. Image of the final universal PCR products by 3% Metaphor agarose gel electrophoresis. Each reaction was performed on a different amount of starting human genomic DNA, as labeled at the top of the figure. The expected smear of products is seen in the lanes that contained between 900 ng and 20 ng DNA. The gel image demonstrates that the reaction generates the expected products when as little as 20 ng of genomic DNA is used. A faint smear is visible in the lane that started with 1.6 ng in images taken at higher exposure.
FIG. 9A-B depicts two graphs showing the bisulfite method performance. (FIG. 9A) Number of sequencing reads per promoter for all 94 targeted promoters, order by length in base-pairs (bp) on the x-axis. Longer promoter amplicons yield fewer sequencing reads (length bias), but 87 amplicons (93%) have coverage within 10 fold of the median coverage (444 reads). The abundance of each promoter ranged from 10 to 5114 reads. (FIG. 9B) Histogram of the pair-wise squared correlation coefficients for the number of reads per promoter for all 48 samples. The mean correlation coefficient is 0.91, indicating that the number of reads per promoter is highly reproducible across patient samples.
FIG. 10 depicts an illustration of methylation at the H19 imprinted locus. Data from four patients who were germline heterozygous for a SNP (rs2251375) in this locus. The sequencing reads are aligned as rows in each panel. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. The position of the SNP is indicated by the red and white column, where a red base indicates reads from the G allele, and a white base indicates reads from the T allele. The percent of reads for each patient that are from the G allele is listed below the patient identifier for each sample. As expected for an imprinted locus, methylation is observed on one allele in both the tumor (left panels) and adjacent normal tissue (right panels) for each patient. Both alleles and both methylated and unmethylated molecules were amplified and sequenced efficiently from this locus in all samples.
FIG. 11A-D depicts an illustration of four promoters that exhibit tumor specific methylation. Sequencing reads from all patients for each type of tissue are grouped together in panels; breast tumors, adjacent normal breast tissues, colon tumors, and adjacent normal colon tissues. The sequencing reads are aligned as rows in each panel, and grouped by patient. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. FIG. 11A ICAM5 promoters exhibit colon and breast tumor specific methylation. FIG. 11B LAMA1 promoters exhibit colon and breast tumor specific methylation. FIG. 11C KCNQ5 promoters exhibit colon tumor specific methylation. FIG. 11D CLSTN2 promoters exhibit colon tumor specific methylation.
FIG. 12 depicts an illustration of allelic tumor specific methylation. Data from six patients who are germline heterozygous for a SNP (rs2854744) in IGFBP3 promoter. The sequencing reads are aligned as rows in each panel. Each base in the read is color coded to indicate the sequence, yellow indicates a methylated cytosine, blue indicates all other bases. The position of the SNP is indicated by the red and white column, where red indicates reads from the A allele, and the C allele is indicated by yellow, if methylated, or white, if unmethylated and converted to a T. Patient ‘Breast 8’ is unmethylated on both alleles in both the tumor (left column) and normal tissue (right column). Patients ‘Breast 4’ and ‘Colon 6’ display tumor-specific methylation on only one allele, and the methylated allele differs between them. Patients ‘Colon 7’ and ‘Colon 12’ display tumor specific methylation on both alleles. Patient ‘Colon 12’ displays different patterns of methylation on each allele in the tumor.
FIG. 13 depicts a schematic of nucleic acid patch PCR with ends defined by oligo-directed FokI digestion. FokI-directing DNA oligonucleotides anneal upstream and downstream of target nucleic acid sequence in genomic DNA. These oligonucleotides contain a FokI restriction endonuclease recognition sequence, which directs FokI digestion of genomic DNA, defining the ends of the PCR template. Nucleic acid patch oligonucleotides are then annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers. The universal primers are then ligated to the amplicons. The universal primer on the 3′ end of the amplicon is modified with a 3 carbon spacer that protects the selected amplicon from the final exonuclease reaction that degrades nonspecific products. The selected amplicons are then amplified together simultaneously by PCR with universal primers.
FIG. 14 shows an image of the agarose gel electrophoresis of the final Universal PCR products of nucleic acid patch PCR with ends defined by oligo-directed FokI digestion. The first lane contains Low Molecular Weight Ladder (NEB), with band sizes denoted on the left. The second lane contains the full reaction and a smear of products in the expected size range is achieved. The remaining lanes are each missing a component of the reaction, demonstrating that all components of the reactions (except Tween) are required to obtain the expected products.
FIG. 15A-G depicts schematic illustrations of various embodiments of the invention. FIG. 15A depicts a multiplex PCR reaction. FIG. 15B depicts an embodiment where the amplified targets from the PCR reaction in FIG. 15A may be trimmed so the ends of the target regions become internal to the PCR primer sequences. FIG. 15C depicts a restriction enzyme reaction creating nucleic acid sequences with defined ends. FIG. 15D depicts an embodiment where oligonucleotides are used to direct Type IIs restriction enzymes to cut at specific sites in the nucleic acid template. This is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a guide for digestion by the type IIs restriction endonuclease enzyme. FIG. 15E depicts single strand specific exonuclease enzyme digestion of nucleic acid templates protected by locus-specific oligonucleotides to define ends of the nucleic acid template. This is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as protection against digestion by the single strand specific exonuclease enzymes. FIG. 15F depicts the ligation of universal primer sequences to nucleic acid sequences. This is facilitated by upstream and downstream nucleic acid patch oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a patch between the desired sequence and upstream and downstream universal primers to be ligated. FIG. 15G depicts PCR amplification for 454 sequencing. The primers for the PCR amplification may be complementary to the upstream and downstream universal primer nucleotide sequences (black segments of the primers). Additionally, the PCR primers may be coupled to nucleic acid sequence barcodes (white segments of the primers). The barcodes may be internal to the primer sequence.



DETAILED DESCRIPTION OF THE INVENTION
PCR amplifies specific nucleic acid sequences through a series of manipulations including denaturation, annealing of oligonucleotide primer pairs, and extension of the primers with DNA polymerase. These steps can be repeated many times, potentially resulting in large amplification of the number of copies of the original target sequence. Multiplex PCR is a variant of PCR that enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. However, current multiplex PCR methods are hampered by the amplification of mispriming events and inter-primer interactions that prevent amplification, as more primer pairs are used.
The present invention provides a method of multiplex PCR that affords a high level of specificity. The method also allows for parallel sequencing of multiple PCR amplification samples in a single sequencing run. Additionally, the invention provides uses for the method. Each is described in more detail below.
I. Nucleic Acid Patch PCR Method
Generally speaking, the method comprises defining the ends of at least two nucleic acid sequences, annealing upstream and downstream nucleic acid patches to each nucleic acid sequence, annealing an upstream and a downstream universal primer to each patch, and subsequently ligating the universal primers to each nucleic acid sequence. The resulting modified nucleic acid sequences may be amplified using primer sequences wherein each primer comprises a nucleic acid sequence tag specific for the sample, and a nucleic acid sequence to prime the sequencing reaction.
(a) Nucleic Acid Template
A method of the invention may be used to amplify nucleic acid sequences. Usually, the nucleic acid sequences may be found in a nucleic acid template. A nucleic acid template may be from any sample that contains nucleic acid molecules. The nucleic acid template may be from humans, animals, plants, microorganisms or viruses. In preferred embodiments, the nucleic acid template is from a human sample. The sample may be fresh, from archeological or forensic samples, or from preserved samples such as paraffin-embedded tissue. The sample may be a solid tissue or a physiological fluid, such as blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, lymphatic fluid, mucous, synovial fluid, peritoneal fluid, or amniotic fluid. Nucleic acid templates may be prepared from the sample using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor). Alternatively, the sample containing the nucleic acid template may be used directly.
The nucleic acid template may be DNA, RNA, or a complementary DNA (cDNA) sequence that is synthesized from a mature messenger RNA. If the nucleic acid template is RNA, the RNA may be reverse transcribed to DNA using methods well known to persons skilled in the art. In a preferred embodiment, the nucleic acid template is DNA.
In some embodiments, suitable quantities of nucleic acid template for the invention may be 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 μg or less. In preferred embodiments, suitable quantities of nucleic acid template for the invention may be 1000, 900, 675, 450, 225, 112, 70, 50, 20, 1.6, 0.8, 0.4 ng or less.
In some embodiments, the nucleic acid template may be treated to prepare the template for specific applications of the invention. In one embodiment, the nucleic acid template may be treated with bisulfite to determine the pattern of methylation. Nucleic acid templates may be treated with bisulfite using methods well known to those of skill in the art, and may be performed using commercially available reagents, following manufacturer's protocols, such as by using the EZ DNA Methylation-Gold KitTM (Zymo Research), the ImprintTM DNA Modification Kit (Sigma), or the like.
(b) Creation of Nucleic Acid Sequences with Defined Ends
The invention encompasses methods for the creation of nucleic acid sequences with defined ends. As used herein, the phrase “defined ends” refers to a nucleic acid sequence where both the 5′ and 3′ end of the sequence is known. Generally speaking, at least three, four, five, six, seven, or more than seven bases of the sequence are known. Non-limiting examples of methods for creating defined ends may include amplification (such as multiplex amplification), restriction endonuclease digestion, single strand specific exonuclease degradation, or triplex formation and cleavage. These methods are described in more detail below.
i. Multiplex Amplification from a Nucleic Acid Sample
Creating defined ends by multiplex amplification may consist of a PCR reaction using primer pairs for desired targets on the nucleic acid template. An exemplary example of a multiplex PCR reaction is depicted in FIG. 15A. Components of the multiplex PCR amplification reaction may include the nucleic acid sequence to be amplified (template; see section I(a) above), one or more primer pairs for delineating the target nucleic acid sequence on the template to be amplified (described below), one or more nucleotide polymerase (described below), deoxynucleotides, and salts and buffers essential for optimal activity of the polymerases in the reaction.
A. Primers
In a method for creating defined ends, the oligonucleotide PCR primers may be typically synthesized using the four naturally occurring deoxynucleotides dATP, dTTP, dCTP and dGTP. In some embodiments of this invention, oligonucleotide primers may also incorporate natural or synthetic deoxynucleotide analogs not normally present in DNA. Incorporation of nucleotide analogs, depicted as “x” in the diagram above, allows for the oligonucleotide primers to be selectively removed (see section (b) below) after amplification of the target nucleic acid. In some embodiments of the invention, a primer may be used such that, at one or more positions of the primer, one or more of the four deoxyribonucleotides in the primer may be replaced with one or more nucleotide analogs. Primers with nucleotide analogs located throughout the primer may also be used. In one preferred embodiment, primers may have one of the deoxynucleotides replaced with a nucleotide analog. In another preferred embodiment, 25%, 30%, 35% 40%, 50%, 60%, 70%, 80%, 90% or 100% of either dATP, dTTP, dCTP or dGTP in the primers may be replaced with a nucleotide analog. In yet another preferred embodiment, the nucleotide analog may be at the 3′-terminus of the primer.
PCR primers may be designed using standard primer design computer software techniques known to individuals skilled in the art. The variables considered during PCR primer design may include primer length, GC pair content, melting temperature, and size of the target nucleic acid amplified by the primer pair. Generally speaking, primers should not form hairpin structures or self- or hetero-primer pairs, but in some embodiments a primer forming a hairpin structure may be used. In a preferred embodiment, primers may comprise a sequence of 15, 20, 25, 30, 35, 40, 45, 50 or more bases complementary to a portion of a template. In another preferred embodiment, the primer melting temperature may be 50, 55, 60, 65, 70 or 75° C. In a preferred embodiment, the primer melting temperature may be 61, 62, 63, 64, 65, 66 or 67° C. In one embodiment, the melting temperature of each primer of the primer pair may be the same. In another embodiment, the melting temperature of each primer of the primer pair may be different for each primer. In yet another embodiment, the difference in melting temperatures between each primer of the primer pair may be 1, 2, 3, 4, 5, 6, 7, 8, 9° C. or more. In another preferred embodiment, the maximum difference in melting temperature between primer pairs may be 5° C. In a preferred embodiment, the GC content of primer may be 10, 20, 30, 40, 50, 60, 70 or 80%. In yet another preferred embodiment the primer pair may be designed to amplify nucleic acid target products that may be 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or more base pairs in length.
B. Nucleotide Polymerases
In one embodiment of a method for creating defined ends, the nucleotide polymerase may be a DNA polymerase. In another embodiment, the nucleotide polymerase may be a thermostable polymerase. In a preferred embodiment, the nucleotide polymerase may be a thermostable DNA polymerase. A thermostable polymerase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each PCR cycle. Non-limiting examples of thermostable polymerases may include polymerases isolated from the thermophilic bacteria Thermus aquaticus (Taq polymerase), Thermus thermophilus (Tth polymerase), Thermococcus litoralis (Tli or VENTTM polymerase), Pyrococcus furiosus (Pfu or DEEPVENTTM polymerase), Pyrococcus woosii (Pwo polymerase) and other Pyrococcus species, Bacillus stearothermophilus (Bst polymerase), Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma acidophilum (Tac polymerase), Thermus rubber (Tru polymerase), Thermus brockianus (DYNAZYMETM polymerase) Thermotoga neapolitana (Tne polymerase), Thermotoga maritime (Tma) and other species of the Thermotoga genus (Tsp polymerase), and Methanobacterium thermoautotrophicum (Mth polymerase). The PCR reaction may contain more than one thermostable polymerase enzyme with complementary properties leading to more efficient amplification of target sequences. For example, a nucleotide polymerase with high processivity (the ability to copy large nucleotide segments) may be complemented with another nucleotide polymerase with proofreading capabilities (the ability to correct mistakes during elongation of target nucleic acid sequence), thus creating a PCR reaction that can copy a long target sequence with high fidelity. The thermostable polymerase may be used in its wild type form. Alternatively, the polymerase may be modified to contain a fragment of the enzyme or to contain a mutation that provides beneficial properties to facilitate the PCR reaction. In one embodiment, the thermostable polymerase may be Taq polymerase. Many variants of Taq polymerase with enhanced properties are known and include AmpliTaqTM, AmpliTaqTM Stoffel fragment, SuperTaqTM, SuperTaqTM plus, LA TaqTM, LApro TaqTM, and EX TaqTM. In a preferred embodiment, the thermostable polymerase used in the multiplex amplification reaction of the invention is the AmpliTaq Stoffel fragment.
C. PCR Reaction Conditions
Buffer conditions for PCR reactions are known to those of ordinary skill in the art. PCR buffers may generally contain about 10-50 mM Tris-HCl pH 8.3, up to about 70 mM KCl, about 1.5 mM or higher MgCl2, to about 50-200 μM each of dATP, dCTP, dGTP and dTTP, gelatin or BSA to about 100 μg/ml, and/or non-ionic detergents such as Tween-20 or Nonidet P-40 or Triton X-100 at about 0.05-0.10% v/v. In some embodiments, betaine may be added to the PCR reactions at about 0.25 to about 1 M. An example of a detailed description of buffer conditions may be found in Example 2.
In some embodiments, the multiplex PCR reaction may contain 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 or more primer pairs. Not all primer pairs will amplify targets with the same efficiency. In some embodiments, PCR primer pairs with similar amplification efficiency may be pooled in separate multiplex PCR reactions to have better representation of all targets. These PCR reactions may be combined after amplification.
In other embodiments, PCR amplification may be performed at a uniform temperature (isothermal PCR). Examples of isothermal PCR methods may include the ramification amplifying method and the helicase-dependent amplification method. In a preferred embodiment of the invention, PCR amplification may be by thermal cycling between a high temperature to melt the nucleic acid strands, a lower temperature to anneal the primers to the target nucleic acid, and an intermediate temperature compatible with the nucleic acid polymerase to elongate the nucleic acid sequence. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C. or more. In a preferred embodiment, the annealing temperature may be 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72° C. In yet another embodiment, the elongation temperature may be 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C. or more. In a preferred embodiment, the elongation temperature may be 70, 71, 72, 73, 74, 75, 80° C. or more.
In certain embodiments, the PCR reaction may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the melting temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the annealing temperature for about 5 to about 60 seconds. In a preferred embodiment, the PCR reaction may be incubated at the annealing temperature for about 30 seconds. In some embodiments, the PCR reaction may be incubated at the elongation temperature for about 1 to about 10 minutes. In a preferred embodiment, the PCR reaction may be incubated at the elongation temperature for about 6 minutes. In some embodiments, the PCR reaction is pre-incubated at the melting temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes before cycling between the melting, annealing and elongation temperatures. In a preferred embodiment, the PCR reaction may be pre-incubated at the melting temperature for about 2 minutes.
In several embodiments, the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or more times. In a preferred embodiment, the PCR reactions may be cycled between the melting, annealing and elongation temperatures 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.
D. Trimming Amplicons
In some embodiments of the invention, the amplified targets from the PCR reaction described above may be trimmed so the ends of the target regions become internal to the PCR primer sequences as depicted in FIG. 15B. The extent of the trimming may generally be defined by synthetic nucleotide analogs incorporated into the primer pairs described above. Treatments that specifically remove synthetic nucleotide analogs have been devised and are well known to those skilled in the art.
In certain embodiments, oligonucleotides containing 5-bromodeoxyuridine (BdUR) or 5-bromodeoxycytidine (BrdC) may be used as the primers of the invention. Primers containing BdUR may be degraded upon exposure to light. In other embodiments, the deoxyinosine may be incorporated into primers of the invention. Primers containing deoxyinosine may be degraded using Endonuclease V, an enzyme that recognizes and cleaves the sugar phosphate backbone at the deoxyinosine residue.
In other embodiments, the base of the synthetic nucleotide is first specifically removed, leaving an apurinic or apyrimidinic site (AP site) and an intact sugar-phosphate backbone. The sugar-phosphate backbone is then cleaved at the AP site, generating a nick in the target, which dictates the nucleic acid sequence to be removed by exonuclease enzymes. In preferred embodiments of the invention, the base of the synthetic nucleotide analog is removed with a DNA glycosylase enzyme. DNA glycosylases are a family of enzymes that can remove the base of some nucleotide analogs. Some examples of nucleotide analogs that may be incorporated into primers and that are substrates for glycosylase enzymes may include deoxyuridine, deoxy-7-methylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3-methyladenosine, deoxyinosine, 5-methyl-deoxycytidine, O-6-methyl-deoxyguanosine, 5-iodo-deoxyuridine, 8-oxy-deoxyguanine, and 1,N6-ethenoadenine. Glycosylase enzymes that remove bases from nucleotide analogs incorporated into target nucleic acid sequences may include uracyl DNA glycosylase, 7-methylguanine-DNA glycosylase, 5,6-dihydroxythymidine glycosylase, 3-methyladenine glycosylase, hypoxanthine DNA N-glycosylases, 8-oxoguanine-DNA glycosylase, and alkylpurine-DNA-N-glycosylase. In a preferred embodiment, the nucleotide analog may be deoxyuridine. In another preferred embodiment, the DNA glycosylase enzyme may be uracil DNA glycosylase.
In some embodiments, treatments that cleave AP sites may include, but are not limited to, heat, alkaline hydrolysis, tripeptides such as Lys-Trp-Lys and Lys-Tyr-Lys, AP endonucleases such as endonuclease III, endonuclease IV, endonuclease VI, endonuclease VIII, phage T4 UV endonuclease, and the like. In a preferred embodiment, the treatment is endonuclease VIII.
After removing primers from amplified target nucleic acid sequences, the resulting single strand overhanging nucleic acid sequence at the 3′ termini may be removed using an enzyme with a 3′ to 5′ single stranded exonuclease activity as depicted in the diagram above. Commonly used 3′ to 5′ exonucleases that remove single stranded nucleic acids may include exonuclease I and exonuclease VII. In a preferred embodiment of the invention, the exonuclease is exonuclease I.
After trimming the ends of the amplified target nucleic acids, other manipulations that prepare the reactions for subsequent steps may be performed. For example, removal of unincorporated nucleotides might be required. In some embodiments, this may be accomplished by physical means such as precipitation, filtration, and chromatography. In other embodiments, the unincorporated nucleotides may be diluted to a concentration where they would not interfere in later steps. In preferred embodiments, the unincorporated nucleotides may be removed using enzymes such as apyrase, an ATP diphosphohydrolase that catalyses the removal of the gamma phosphate from ATP and the beta phosphate from ADP.
ii. Restriction Endonuclease Enzymes
In another embodiment, restriction endonuclease enzymes may be used to create nucleic acid sequences with defined ends. Suitable restriction endonuclease enzymes may include type I, type II, type III or type IV restriction endonuclease enzymes. Generally speaking, the restriction enzyme used should have recognition sites that flank, and not bisect, the desired nucleic acid sequence. In some embodiments, the restriction endonuclease enzymes may be type I restriction endonuclease enzymes. Non-limiting examples of Type I restriction endonuclease enzymes may include CfrI, Eco377I, EcoAI, EcoDXXI, EcoKI, Eco124I, KpnAI, and StySPI. In other embodiments, the restriction endonuclease enzymes may be type II restriction endonuclease enzymes. Type II restriction endonuclease enzymes suitable for the methods of the invention may be a restriction endonuclease enzyme of type IIB, type IIE, type IIF, type IIG, type IIM, type IIS, or type IIT. In certain embodiments, Type III restriction endonuclease enzymes may be suitable for the methods of the invention. Non-limiting examples of Type III restriction endonuclease enzymes are known in the art. In alternative embodiments, the restriction endonuclease enzymes may be Type IIS restriction endonuclease enzymes. Non-limiting examples of Type IIS restriction endonuclease enzymes may include FokI, HgaI, EciI, BceAI, BbvI, BtgZI, BsmFI, BpmI, and BsgI. Other restriction endonuclease enzymes are known in the art. For instance, additional non-limiting examples may be found at http://rebase.neb.com/cgi-bin/azlist?re1, http://rebase.neb.com/cgi-bin/azlist?re2, http://rebase.neb.com/cgi-bin/azlist?re3, or http://rebase.neb.com/cgi-bin/azlist?re4.
The restriction endonuclease enzyme cut sites may be used to define the ends of nucleic acid templates. An exemplary example of a restriction enzyme reaction creating nucleic acid sequences with defined ends is depicted in FIG. 15C. Components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more restriction endonucleases, and salts and buffers essential for optimal activity of the enzymes in the reaction. The restriction enzyme reaction may be prepared using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
In some embodiments, oligonucleotides may be used to direct Type IIs restriction enzymes to cut at specific sites in the nucleic acid template. As depicted in FIG. 15D, this is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a guide for digestion by the type IIs restriction endonuclease enzyme. Thus, components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more restriction endonucleases, the oligonucleotides directing the restriction endonuclease cut sites (described below), and salts and buffers essential for optimal activity of the enzymes in the reaction.
A. Oligonucleotides Directing Type IIs Restriction Endonuclease Enzymes
The upstream and downstream restriction enzyme-directing oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described in section I(b)iA above. In some preferred embodiments, the 5′ ends of the upstream restriction enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequence (e.g. the segment parallel to the genomic DNA in the diagram above), and may be concatenated at the 3′ end of the oligonucleotides to double-stranded nucleotide sequences encoding type IIs restriction enzymes. In other preferred embodiments, the 3′ ends of the downstream restriction-enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequences (e.g. the segment parallel to the genomic DNA in the diagram above), and may be concatenated at the 5′ end of the oligonucleotides to double-stranded nucleotide sequences encoding type IIs restriction enzymes.
B. Annealing and Digestion Reaction Conditions
Annealing of the restriction enzyme-directing oligonucleotides to the nucleic acid templates may generally be performed before addition of the restriction enzyme for digestion. In addition to the nucleic acid template, annealing reactions may generally contain about 1 pM to about 500 nM of each restriction enzyme-directing oligonucleotide, and about 0.01 to about 0.9% Tween80. An example of a detailed description of buffer conditions may be found in Example 8.
In some embodiments, annealing of the restriction enzyme-directing oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the restriction enzyme-directing oligonucleotides to target nucleic acid sequences. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more. In a preferred embodiment, the annealing temperatures may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, or 52° C.
In other embodiments, the annealing reactions may be incubated at the melting temperature for about 5 to about 30 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes. In some embodiments, the annealing reactions may be incubated at the annealing temperature for about 1 to about 10 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes.
After annealing the restriction enzyme-directing oligonucleotides to the template, the type IIs restriction enzyme may be added, and the restriction enzyme reaction may be prepared using methods well known to those of skill in the art (see, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual,” 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor).
iii. Single Strand Specific Exonuclease Degradation
Single strand specific exonuclease enzyme digestion of nucleic acid templates protected by locus-specific oligonucleotides may be used to define ends of the nucleic acid template. As depicted in FIG. 15E, this is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as protection against digestion by the single strand specific exonuclease enzymes. Thus, components of the exonuclease reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more single strand specific exonuclease enzymes (described below), the oligonucleotides protecting the nucleic acid template (described below), and salts and buffers essential for optimal activity of the exonucleases in the reaction.
Non-limiting examples of single strand specific exonuclease enzymes suitable for the methods of the invention may be exonuclease VII, exonuclease I, RecJ exonuclease, or TerminatorTM 5′-Phosphate-Dependent Exonuclease (Epicentre Biotechnologies). The upstream and downstream oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described in (b)i.A. above.
Annealing of the protecting oligonucleotides to the nucleic acid templates may generally be performed before addition of the exonuclease enzymes. In addition to the nucleic acid template, annealing reactions may generally contain about 1 pM to about 500 nM of each oligonucleotide. In some embodiments, annealing of the oligonucleotides may be performed by melting the nucleic acid strands at a high temperature, followed by a lower temperature suitable for annealing the protecting oligonucleotides to target loci. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the annealing temperatures may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55° C. or more. In a preferred embodiment, the annealing temperatures may be about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 50, 51, or 52° C.
In some embodiments, the annealing reactions may be incubated at the melting temperature for about 5 to about 30 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 minutes. In some embodiments, the annealing reactions may be incubated at the annealing temperature for about 1 to about 10 minutes. In a preferred embodiment, the annealing reactions may be incubated at the melting temperature for about 1, 2, 3, 13, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes. After annealing of the protecting oligonucleotides, the exonuclease enzymes may be added for digestion.
iv. Triplex Formation and Cleavage by Endonucleases
The ability of some nucleic acid recombination proteins to direct the formation of triplex nucleic acid structures may be used to create defined ends of a nucleic acid sequence. Triplex DNA structures are induced at specific loci by incubating nucleic acid templates with locus-specific oligonucleotides that have been coated with the recombination protein. The triplex structure then produces a single stranded region of nucleic acid available for cleavage by single strand specific endonucleases. Thus, components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more recombination proteins, the recombination protein-coated locus-specific oligonucleotides, the endonuclease proteins, and salts and buffers for optimal activity of the enzymes. Non-limiting examples of recombination proteins may include RecA of Escherichia coli, or any homologous recombination protein capable of inducing formation of triplex DNA structure. Non-limiting examples of single strand specific endonucleases may include S1 and BAL1 endonucleases.
(c) Nucleic Acid Patch PCR
One aspect of the invention is the ligation of universal primer sequences to nucleic acid sequences. As depicted in FIG. 15F, this is facilitated by upstream and downstream nucleic acid patch oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a patch between the desired sequence and upstream and downstream universal primers to be ligated. Thus, nucleic acid patch ligation reactions contain the target sequences, the upstream and downstream universal primers to be ligated, the upstream and downstream nucleic acid patch oligonucleotides to guide the specific ligation of the universal primers, and the enzymes and other components needed for the ligation reaction. In preferred embodiments, target sequences may be nucleic acid sequences with defined ends as described above.
i. Universal Primers
The upstream and downstream universal primers may be designed using primer length, GC pair content and melting temperature criteria as described in I(a) above. In some embodiments, the downstream universal primer may be modified to facilitate further steps of the invention. In a specific embodiment, the downstream universal primer may be modified with a 5′ phosphate group to enable ligation of the downstream universal primer to the amplicon. In other specific embodiments, the 3′ end of the downstream universal primer may be modified for protection against exonuclease digestion. Modifications at the 3′ end may be introduced at the time of synthesis or after synthesis through chemical means well know to those of skill in the art. Modifications may be 3′ terminal or slightly internal to the 3′ end. Some examples of modifications that make nucleic acid sequences exonuclease resistant include, but are not limited to, locked nucleic acids (LNA's), 3′-linked amino groups, 3′ phosphorylation, the use of a 3′-terminal cap (e.g., 3′-aminopropyl modification or by using a 3′-3′ terminal linkage), phosphorothioate modifications, the use of attachment chemistry or linker modification such as Digoxigenin NHS Ester, Cholesteryl-TEG, biotinylation, thiol modifications, or addition of various fluorescent dyes and spacers such as C3 spacer. In a preferred embodiment, the downstream universal primer is protected from exonuclease digestion by a C3 spacer.
ii. Nucleic Acid Patch Primers
In some embodiments, an upstream and a downstream nucleic acid patch oligonucleotide may be designed for each amplicon (see diagram above). In some preferred embodiments, the 5′ ends of the upstream nucleic acid patch oligonucleotides may be complementary to sequences in the amplicons (grey segment of upstream nucleic acid patch oligonucleotide in diagram above), and may be concatenated to upstream nucleotide sequences complementary to the upstream universal primer sequence on the 3′ end (black segment of upstream nucleic acid patch oligonucleotide in diagram above). In other preferred embodiments, the 3′ ends of the downstream nucleic acid patch oligonucleotides may be complementary to downstream sequences in the amplicons (grey segment of downstream Nucleic acid patch oligonucleotide in diagram above), and may be concatenated to nucleotide sequences complementary to the downstream universal primer sequence on the 5′ end (black segment of downstream nucleic acid patch oligonucleotide in diagram above).
iii. Ligation of Universal Primers
In some embodiments, the universal primers may be ligated to nucleic acid sequences. In a process similar to a PCR amplification reaction, multiple cycles of heating and cooling may be used to melt the target nucleic acid sequence, anneal the nucleic acid patch and universal primers, and ligate the universal primers to target nucleic acid sequences.
In some embodiments of the invention, the universal primers of the invention may be ligated to the target nucleic acids using a DNA ligase. The ligase may be theromostable. In preferred embodiments, the ligase is a thermostable DNA ligase. A thermostable DNA ligase is an enzyme that is relatively stable to heat and eliminates the need to add enzyme prior to each ligation cycle. Non-limiting examples of theromostable DNA ligases may include Ampligase® Thermostable DNA Ligase, Taq DNA Ligase from Thermus aquaticus, Tfi DNA ligase from Thermus filiformis, Tth DNA ligase from Thermus thermophilus, Thermo DNA ligase, Pfu DNA ligase from Pyrococcus furiosus, and thermostable DNA ligase from Aquifex pyrophilus. The thermostable polymerase may be used in its wild type form, modified to contain a fragment of the enzyme, or to contain a mutation that provides beneficial properties to facilitate the ligation reaction. In a preferred embodiment, the thermostable ligase is Ampligase®.
iv. Ligation Reaction Conditions
Ligation reactions may generally contain about 1 pM to about 500 nM of each nucleic acid patch oligo, about 1 pM to about 500 nM of each universal primer, about 3, 4, 5, 6, 7, or 8 units of Ampligase®, and 1× Ampligase Reaction Buffer. An example of a detailed description of buffer conditions may be found in Example 2.
In some embodiments, ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a sequence of 1, 2, 3, 4 or 5 lower temperatures to anneal the nucleic acid patch oligonucleotides to the target nucleic acid, and a temperature compatible with the ligase to ligate the nucleic acid sequence. In a preferred embodiment, ligation reactions may be performed by thermal cycling between a high temperature to melt the nucleic acid strands, a first lower temperature to anneal the nucleic acid patch oligonucleotides to the target nucleic acid, a second lower temperature to anneal the universal primers to the nucleic acid patch oligonucleotides, and a temperature compatible with the ligase to ligate the nucleic acid sequence. In one embodiment, the melting temperatures may be about 85, 86, 87, 88, 89, 90, 95, or 100° C. In a preferred embodiment, the melting temperature may be about 90, 91, 92, 93, 94, 95, 96, 97 or 98° C. In another embodiment, the Nucleic acid patch oligonucleotide annealing temperatures may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75° C. or more. In a preferred embodiment, the nucleic acid patch oligonucleotide annealing temperatures may be about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 70, 71, or 72° C. In another embodiment, the ligation temperature may be about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80° C. or more. In a preferred embodiment, the ligation temperature may be about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70° C. or more.
In some embodiments, the ligation reactions may be incubated at the melting temperature for about 5 to about 60 seconds. In a preferred embodiment, the ligation reactions may be incubated at the melting temperature for about 30 seconds. In some embodiments, the ligation reactions may be incubated at the nucleic acid patch oligonucleotide annealing temperature for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more minutes. In a preferred embodiment, the reactions may be incubated at the nucleic acid patch oligonucleotide annealing temperature for about 2 minutes. In some embodiments, the ligation reactions may be incubated at the universal primer annealing temperature for about 30 seconds to about 5 minutes. In a preferred embodiment, the ligation reactions may be incubated at the universal primer annealing temperature for about 1 minute. In some embodiments, the ligation reactions may be incubated at the ligation temperature for about 30 seconds to about 5 minutes. In a preferred embodiment, the ligation reactions may be incubated at the ligation temperature for about 1 minute. In some embodiments, the reactions may be pre-incubated at the melting temperature for about 5, 6, 7, 8, 9, 10, 15, 20 or 25 minutes before cycling between the melting, annealing and ligation temperatures. In a preferred embodiment, the ligation reactions may be pre-incubated at the melting temperature for about 15 minutes.
In some embodiments, the ligation reactions may be cycled between the melting, annealing and ligation temperatures about 10, 50, 100, 150, 200 or more times. In a preferred embodiment, the ligation reactions may be cycled between the melting, annealing and elongation temperatures about 100 times.
(d) Degrade Mispriming Products and Genomic DNA
In some embodiments, exonucleases may be added to the ligation reaction at the completion of the reaction to degrade mispriming products of the multiplex PCR reaction or genomic DNA. In preferred embodiments, exonucleases may be 3′ to 5′ exonucleases. Exonucleases may be single stranded or double stranded exonucleases. Non-limiting examples of exonucleases suitable for this step of the reaction may include exonuclease I, exonuclease III and mung bean nuclease. One or more exonucleases may be added. In a preferred embodiment, the exonucleases may be exonuclease I and III.
(e) Sample-Specific Barcode PCR and Sequencing of Nucleic Acid Patch Amplicons
In some aspects of the invention, nucleic acid samples may be sequenced. In some embodiments, the nucleic acids sequenced may be the amplicons prepared in (a), (b) and (c) above. Sequencing techniques suitable for the invention may be high throughput. High throughput sequencing techniques may include techniques based on chain termination, pyrosequencing (sequence by synthesis), or sequencing by ligation and are well known to those of skill in the art. In some embodiments, high throughput sequencing techniques like true single molecule sequencing (tSMS) may not require amplification of target nucleotide sequences. In preferred embodiments, sequencing may be performed using high throughput sequencing techniques that involve in vitro clonal amplification of the target nucleotide sequence. Non-limiting examples of high throughput sequencing techniques that involve amplification may include solid-phase PCR in polyacrylamide gels, emulsion PCR, rolling-circle amplification, bridge PCR, BEAMing (beads, emulsions, amplification and magnetics)-based cloning on beads, massively parallel signature sequencing (MPSS) to generate clonal bead arrays. In a preferred embodiment, the amplicons may be sequenced using PCR techniques as exemplified by 454 SequencingTM. The PCR amplification for 454 sequencing may be as depicted in FIG. 15G.
In some embodiments, the PCR may use primers complementary to the universal primer sequences described in section I(c)i above, and depicted as black segments in the diagram. In other embodiments, the PCR primers may be coupled to nucleic acid sequences for sequencing (grey segments of the primers in diagram above). In a preferred embodiment, the primers for the final universal PCR may be tailed to 454 sequencing primers A and B (454 Life Sciences, Branford, Conn.). In other embodiments, the primers for the PCR amplification may be complementary to the upstream and downstream universal primer nucleotide sequences ligated in FIG. 15G (black segments of the primers). In additional embodiments, the PCR primers may be coupled to nucleic acid sequence barcodes (white segments of the primers in FIG. 15G). In some embodiments, the nucleic acid barcode may be about 4, 5, 6, 7, 8, 9, 10, or more bases. In a preferred embodiment, the nucleic acid barcode may be about 6 bases. The barcodes may be at the 5′ end, the 3′ end or, as exemplified in FIG. 15G, internal to the primer sequence.
In some embodiments, nucleic acid sequences amplified in the PCR reactions of more than one sample may be pooled for parallel sequencing of nucleic acids prepared in multiple samples. In some embodiments, about 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more samples may be pooled for sequencing.
(f) Specific Embodiments
In a specific embodiment, restriction endonuclease enzymes may be used to create nucleic acid sequences with defined ends. Suitable restriction endonuclease enzymes may be as described in Section I(b)ii. In a specific embodiment, the restriction endonuclease enzyme may be Type II restriction endonuclease enzymes. In another specific embodiment, the restriction endonuclease enzyme may be Type IIS restriction endonuclease enzymes.
In a specific embodiment, oligonucleotides may be used to direct Type IIS restriction enzymes to cut at specific sites in the nucleic acid template. This is facilitated by upstream and downstream oligonucleotides that anneal upstream and downstream of the target nucleic acid sequences and serve as a guide for digestion by the Type IIS restriction endonuclease enzyme. Thus, components of the restriction enzyme reaction may include the nucleic acid sequence to be digested (template; see section I(a) above), one or more restriction endonucleases, the oligonucleotides directing the restriction endonuclease cut sites, and salts and buffers essential for optimal activity of the enzymes in the reaction.
The upstream and downstream restriction enzyme-directing oligonucleotides may be designed using primer length, GC pair content, and melting temperature criteria as described in section I(b)iA above. In a specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may each form a hairpin structure. Specifically, the 5′ ends of the upstream restriction enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequence (e.g. the segment parallel to the genomic DNA in FIG. 13), and may form a hairpin structure at the 3′ end of the oligonucleotides to generate a double-stranded nucleotide sequence recognized by Type IIS restriction enzymes. Further, the 3′ ends of the downstream restriction-enzyme-directing oligonucleotides may be complementary to a portion of the desired nucleic acid sequences (e.g. the segment parallel to the genomic DNA in the diagram above), and may form a hairpin structure at the 5′ end of the oligonucleotides to generate a double-stranded nucleotide sequence recognized Type IIS restriction enzymes.
The upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 10, 15, 20, 25, 30, 35, 40, 45, 50 or more bases complementary to a portion of a template. In a specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 10 or more bases complementary to a portion of a template. In another specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 15 or more bases complementary to a portion of a template. In still another specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 20 or more bases complementary to a portion of a template. In still yet another specific embodiment the upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bases complementary to a portion of a template. In an exemplary embodiment the upstream and downstream restriction enzyme-directing oligonucleotides may comprise a sequence of 15, 16, 17, 18, 19, or 20 bases complementary to a portion of a template. It is not necessary that the upstream and downstream restriction enzyme-directing oligonucleotides comprise the same amount of bases complementary to a portion of a template.
The upstream and downstream restriction enzyme-directing oligonucleotides may comprise 20, 25, 30, 35, 40, 45, 50 or more bases. In a specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise 20 or more bases. In another specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise 25 or more bases. In still another specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise 30 or more bases. In still yet another specific embodiment, the upstream and downstream restriction enzyme-directing oligonucleotides may comprise 35 or more bases. It is not necessary that the upstream and downstream restriction enzyme-directing oligonucleotides comprise the same amount of bases.
The annealing and digestion reaction conditions may be as described in section I(b)iiB and the rest of the method may be as described in sections I(c)-(e).
The specific embodiments described above may be used in a method of detecting short tandem repeats (STRs). As used herein, a STR consists of a unit of about two to about 13 nucleotides repeated up to hundreds of times in a row on the DNA. The method of detecting STRs may be used to measure the number of repeating units and/or measure the length of the STR. According to the specific embodiment described above, upstream and downstream restriction enzyme-directing oligonucleotides anneal to regions on the DNA immediately adjacent to the STR and the methodology disclosed herein may be used to detect the number of repeating units in the STR and/or the length of the STR. Such a method may be useful in the field of forensic science. By immediately adjacent is meant that the oligonucleotides anneal to the base directly next to the STR or about 1 or up to 20 bases upstream and downstream from the STR. For example, the oligonucleotides may anneal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases upstream and downstream from the STR. In a specific embodiment, the oligonucleotides may anneal 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream and downstream from the STR. The upstream oligonucleotide does not have to be the same distance from the STR as the downstream oligonucleotide.
II. Methods of Use
A further aspect of the invention provides uses for the amplification method detailed herein. In some embodiments, a method described herein may be used to detect and discover single nucleotide polymorphisms (SNPs) or mutations. In other embodiments, a method described herein may be used to detect pathogen DNA in a high background of host DNA, detect rare DNA to allow for multiplexed or genome-wide amplification of biomarkers in peripheral samples, or amplify targets from degraded samples to allow for multiplexed or genome-wide amplification. In a specific embodiment, a method described herein may be used to detect rare tumor DNA to allow for multiplexed or genome-wide amplification of biomarkers in peripheral samples such as blood or stool. In yet other embodiments, a PCR method described herein may be used to detect DNA methylation. Other applications that rely heavily on PCR may benefit from higher levels of multiplexing, such as the amplification of all exons or all conserved regions, or the engineered assembly of many DNA fragments simultaneously in synthetic biology experiments, or the analysis of short tandem repeats.
In still other embodiments, the PCR method described herein may be used to detect DNA methylation, detect and/or sequence tumor DNA derived from peripheral samples (blood, stool), amplify all exons in a particular template, or amplify all conserved regions in a particular template. A skilled researcher in the art will appreciate that other methods of use for a method detailed herein may be possible or desirable, and that the methods of use detailed herein are not to be construed as limiting.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
EXAMPLES
The following examples illustrate various iterations of the invention.
Example 1: Nucleic Acid Patch PCR Design
Mispriming events plague standard multiplex PCR reactions as the number of primer pairs increases. Nucleic acid patch PCR was designed to significantly decrease mispriming events, as nucleic acid patch PCR requires four oligonucleotide hybridizations per locus. This results in a more specific amplification than standard multiplex PCR, which requires only two hybridizations per locus. FIG. 1 presents a schematic of the concept of nucleic acid patch PCR.
In the first round of oligonucleotide hybridization, a PCR reaction containing DNA primer pairs for all targets is performed on genomic DNA (FIG. 1A). These DNA primers contain uracil substituted for thymine to facilitate the next step of the process. The PCR is performed for a low number of cycles and serves to define the ends of the target regions. To prepare for the second round of oligonucleotide hybridization, the PCR product generated above is first trimmed to produce a nucleic acid fragment with ends internal to the PCR primer sequences (FIG. 1B). This is accomplished by removing the uracil-containing primers, and trimming the resulting DNA overhangs on the PCR product by an enzyme mix containing uracil DNA glycosylase.
Next, a second round of oligonucleotide hybridization is performed. Nucleic acid patch oligonucleotides are annealed to the target amplicons and serve as a patch between the correct amplicons and universal primers (FIG. 1C). In the third oligonucleotide hybridization, the universal primers are annealed to the nucleic acid patch primers, and then ligated to the amplicons in a reaction containing a thermostable ligase followed by exonucleases I and III. This reaction provides two levels of selection in addition to the oligonucleotide hybridization. First, the thermostable ligase used is sensitive to mismatched bases near the ligation junction (Barany 1991), and second, the exonucleases in the reaction provide an added level of selectively by degrading mispriming products and the genomic DNA. The selected amplicons are protected from the exonuclease in the final reaction by a 3′ modification with a 3-carbon spacer on the universal primer. The selected amplicons are then amplified together simultaneously by PCR with the universal primers (FIG. 1D) for the final round of selection.
The target selection protocol is an addition-only reaction and can be performed in a single tube per sample, making it amenable to automation. To pool and sequence multiple samples, nucleic acid patch PCR is first performed separately for each sample (1 tube per sample). Sample-specific DNA barcodes are then incorporated into the primers used for the final universal PCR by tailing the 5′ end with sample-specific DNA sequences and 454 sequencing primers (FIG. 2). Thus, the first few bases indicate from which sample each read originated.
Example 2: Nucleic Acid Patch PCR and Sequencing of Candidate Genes in Colon Cancer
To demonstrate the multiplexed selection and amplification of exons by nucleic acid patch PCR described in Example 1, single nucleotide polymorphisms (SNPs) and mutations were analyzed in six nucleotide sequences encoding cancer related proteins: tumor protein p53 (TP53); adenomatous polyposis coli (APC); mutL homolog 1, colon cancer, nonpolyposis type 2 (MLH1); retinoblastoma 1 (RB1); breast cancer 1, early onset protein (BRCA1); and von Hippel-Lindau tumor suppressor protein (VHL) (Marsh and Zori 2002). These targets are located across 4 chromosomes, vary in length from 74 bp to 438 bp, and total 21.6 kbp. Oligonucleotide design, conditions of PCR reactions, sequencing and sequence analysis are described below.
Oligonucleotide Design
Human exon sequence plus 150 bp flanking sequence from the March 2006 assembly was downloaded from the UCSC Genome Browser (www.genome.ucsc.edu). The reference sequences (Refseq) representing the six colon cancer related nucleic acids were: NM_000038 (APC), NM_000546 (TP53), NM_000249 (MLH1), NM_000321 (RB1), NM_007304 (BRCA1), and NM_000551 (VHL). The convention that exon numbering for each gene begins with zero was maintained throughout the analysis. Primer3 software (http://frodo.wi.mit.edu/) was then used to select primer pairs flanking the exon. The design was constrained to PCR products between 50-500 bp, primer length 20-36 bp, primer melting temperature (Tm)=61-67° C., where the maximum difference in Tm between primer pairs was 5° C., and the GC content of the primer had to be between 10-80%. Four thousand possible primer pairs were generated per exon. Those primer pairs that ended with a T as the 3′ base were then selected. Oligonucleotide sequences of the PCR primers are listed in TABLE A. All PCR primer oligonucleotides were synthesized by Sigma-Genosys.
A nucleic acid patch oligonucleotide was then designed by extending into the sequence from the PCR primer until the Tm of the nucleic acid patch oligonucleotide was 62-67° C. The selected oligonucleotides were then aligned against themselves using BLASTN software from the Washington University BLAST Archives WUBLAST (http://blast.wustl.edu) to approximate cross reactivity. For each exon, the oligonucleotide sets with the fewest blastn matches to the entire set were chosen. The PCR primer sequence was substituted with a deoxyuridine in place of every deoxythymidine. The nucleic acid patch oligonucleotides were then concatenated with the complement universal primer sequences to result in the appropriate patch sequence. Sequences of the nucleic acid patch oligonucleotides are listed in TABLE B. All nucleic acid patch oligonucleotides were synthesized by Sigma-Genosys.
Two Universal Primer oligonucleotides were synthesized for the ligation reaction, including the Universal Primer 2, which has a 5′ Phosphate and a 3-carbon spacer on the 3′ end. The Universal Primer oligonucleotide sequences were then tailed at the 5′ end with the sample-specific DNA barcodes and 454 Life Sciences A or B oligonucleotide sequence to result in the Final Universal Primer oligonucleotides for normal samples and colon cancer samples. The Universal Primer oligonucleotides for ligation and the Final Universal primer oligonucleotide sequences for normal and colon cancer samples are listed in TABLE C.







TABLE A







Multiplex PCR









SEQ




ID.




NO.
Oligo Name
Sequence












1
000038_00_PCRleft
TCTTAAGAGTTTTGTTTCCTTTACCCCU


 


2
000038_01_PCRleft
CGTGCTTTGAGAGTGATCTGAATTU


 


3
000038_02_PCRleft
TTGTGGTTAAAATGTAAACCTAATATTTCACU


 


4
000038_03_PCRleft
GGTAGAGAAGTTTGCAATAACAACTGAU


 


5
000038_04_PCRleft
AAATAATTTTCTCATGCACCATGACU


 


6
000038_05_PCRleft
TTAAATGAGAATGATTTGACATAACCCU


 


7
000038_06_PCRleft
AAAAAGCCTTGGGCTAAGAAAGCCU


 


8
000038_07_PCRleft
AATGGTCATACTTTTATGATGTATTTAATTGTTU


 


9
000038_08_PCRleft
GCTTTTGGATATTAAAGTCGTAATTTTGTTU


 


10
000038_09_PCRleft
ATTTGTTGATCCACTAAAATTCCGU


 


11
000038_10_PCRleft
TGATTGTCTTTTTCCTCTTGCCCTU


 


12
000038_11_PCRIeft
AAAGCTTGGCTTCAAGTTGTCTTTU


 


13
000038_12_PCRleft
AAAGTGATAGGATTACAGGCGTGAGU


 


14
000038_13_PCRleft
GAAGTTAATGAGAGACAAATTCCAACTCU


 


15
000249_00_PCRleft
CCGTTGAGCATCTAGACGTTTCCU


 


16
000249_01_PCRleft
CCTGTAAGACAAAGGAAAAACACGTTAAU


 


17
000249_02_PCRleft
TGGATTAAATCAAGAAAATGGGAAU


 


18
000249_03_PCRleft
CAGCAGTTCAGATAACCTTTCCCTTU


 


19
000249_04_PCRleft
TGTTGATATGATTTTCTCTTTTCCCCTU


 


20
000249_05_PCRleft
TGGATTCACTATCTTAAGACCTCGCTTU


 


21
000249_06_PCRleft
GGGCTCTGACATCTAGTGTGTGTTU


 


22
000249_07_PCRleft
TCCTTGTGTCTTCTGCTGTTTGTTU


 


23
000249_08_PCRleft
GAGGACCTCAAATGGACCAAGTCU


 


24
000249_09_PCRleft
GGTGATTTCATGACTTTGTGTGAATGU


 


25
000249_10_PCRleft
ATCTTCTGGCCACCACATACACCAU


 


26
000249_11_PCRleft
GCTCCATTTGGGGACCTGTATATCU


 


27
000249_12_PCRleft
GCTCTGTAGAACCAGCACAGAGAAGTU


 


28
000249_13_PCRleft
AGGCTTCTTTGCTTACTTGGTGTCU


 


29
000249_14_PCRleft
TCTCATCCATGTTTCAGGGATTACU


 


30
000249_15_PCRleft
TTGCTCCTTCATGTTCTTGCTTCTU


 


31
000249_16_PCRleft
ATCAAGTAACGTGGTCACCCAGAGU


 


32
000249_17_PCRleft
CAGCAATATTCAGCAGTCCCATTU


 


33
000249_18_PCRleft
ATCAGCCAGGACACCAGTGTATGTU


 


34
000321_00_PCRleft
GAAGTGACGTTTTCCCGCGGU


 


35
000321_01_PCRleft
GATCTTAAAGTATTTAATAATGTTCTTTTTCACAGU


 


36
000321_02_PCRleft
CCATCAGAAGGATGTGTTACAAATATACAGU


 


37
000321_03_PCRleft
AATTCCTTCCAAAGGATATAGTAGTGATTU


 


38
000321_04_PCRleft
TCTTAAAAGAAGATAAATAAAGCATGAGAAAACU


 


39
000321_05_PCRleft
GCACAAAAAGAAACACCCAAAAGAU


 


40
000321_06_PCRleft
CATGCTGATAGTGATTGTTGAATGAAU


 


41
000321_07_PCRleft
GGATGTACAATTGTTCTTATCTAATTTACCACTU


 


42
000321_08_PCRleft
CATGGGGGATTGACACCTCTAACU


 


43
000321_09_PCRleft
AAAATTCTTTAATGAAATCTGTGCCTCU


 


44
000321_10_PCRleft
TTATATGATTTTATGAGACAACAGAAGCATU


 


45
000321_11_PCRleft
AACCACAGTCTTATTTGAGGGAATGU


 


46
000321_12_PCRleft
CGACATTGATTTCTGTTTTTACCTCCU


 


47
000321_14_PCRleft
TGAGCCAAGATTGTGCCATU


 


48
000321_15_PCRleft
AATTATCTGTTTCAGGAAGAAGAACGAU


 


49
000321_16_PCRleft
TGGTTTAACCTTTCTACTGTTTTCTTTGTCU


 


50
000321_17_PCRleft
TTCATTCTGACTTTTAAATTGCCACU


 


51
000321_18_PCRleft
TCTGGGTGTACAACCTTGAAGTGTAU


 


52
000321_19_PCRleft
TCTGGGGGAAAGAAAAGAGTGGU


 


53
000321_20_PCRleft
AAAGAAATAACTCTGTAGATTAAACCTTTCTTTU


 


54
000321_21_PCRleft
TTTCCTTTATAATATGTGCTTCTTACCAGU


 


55
000321_22_PCRleft
TCTTCATGCAGAGACTGAAAACAAAU


 


56
000321_23_PCRleft
TTTGGTATTCCTAATAGTTCAGAATGATGU


 


57
000321_24_PCRleft
CTTTGCCTGATTTTTGACACACCU


 


58
000321_25_PCRleft
AATAGCATAAAGTAAGTCATCGAAAGCAU


 


59
000321_26_PCRleft
TGTCAAATACTAGAATGAAGACCACTGCU


 


60
000546_00_PCRleft
GTCTCAGACACTGGCATGGTGU


 


61
000546_01_PCRleft
CATTTTCAGACCTATGGAAACTGTGAGU


 


62
000546_02_PCRleft
ACAACGTTCTGGTAAGGACAAGGGU


 


63
000546_03_PCRleft
AGGTGCTTACGCATGTTTGTTTCTU


 


64
000546_04_PCRleft
AGTCACAGCACATGACGGAGGTU


 


65
000546_05_PCRleft
TGAGCTGAGATCACGCCACU


 


66
000546_06_PCRleft
CTCCAGAAAGGACAAGGGTGGU


 


67
000546_07_PCRleft
TATCACCTTTCCTTGCCTCTTTCCU


 


68
000546_08_PCRleft
TACTTACTTCTCCCCCTCCTCTGTU


 


69
000546_09_PCRleft
CACCATCTTGATTTGAATTCCCGU


 


70
000551_00_PCRleft
CGAGCGCGTTCCATCCTCU


 


71
000551_01_PCRleft
CCCAAAGTGCTGGGATTACAGGU


 


72
000551_02_PCRleft
AAGCCTCTTGTTCGTTCCTTGTACU


 


73
007304_00_PCRleft
GGTTTGTATTATTCTAAAACCTTCCAAATCTU


 


74
007304_01_PCRleft
TTATTGAGCCTCATTTATTTTCTTTTTCU


 


75
007304_02_PCRleft
GCTCTTAAGGGCAGTTGTGAGATTAU


 


76
007304_03_PCRleft
TGCTGAGTGTGTTTCTCAAACAATTU


 


77
007304_04_PCRleft
TCACAGGTAACCTTAATGCATTGTCTU


 


78
007304_05_PCRleft
TCTTCAGGAGGAAAAGCACAGAACU


 


79
007304_06_PCRleft
TTAACTAGCATTGTACCTGCCACAGU


 


80
007304_07_PCRleft
AAAGGAGAGAGCAGCTTTCACTAACU


 


81
007304_08_PCRleft
TGACAATTCAGTTTTTGAGTACCTTGTU


 


82
007304_09_PCRleft
CCAAAGCAAGGAATTTAATCATTTTGU


 


83
007304_10_PCRleft
ATTTTCTTGGTGCCATTTATCGTTU


 


84
007304_11_PCRleft
TCACTATCAGAACAAAGCAGTAAAGTAGATU


 


85
007304_12_PCRleft
TGATCTCTCTGACATGAGCTGTTTCAU


 


86
007304_13_PCRleft
TGTGTAAATTAAACTTCTCCCATTCCTU


 


87
007304_14_PCRleft
GTAGAACGTGCAGGATTGCTACAU


 


88
007304_15_PCRleft
AAATCCAGATTGATCTTGGGAGTGU


 


89
007304_16_PCRleft
AGCCTTATTAAAGGGCTGTGGCTTU


 


90
007304_17_PCRleft
CTAGGATTACAGGGGTGAGCCACU


 


91
007304_18_PCRleft
ATTTTCCTTCTCTCCATTCCCCTGU


 


92
007304_19_PCRleft
CCTTCATCCGGAGAGTGTAGGGU


 


93
007304_20_PCRleft
TCCTACTTTGACACTTTGAATGCTCTU


 


94
007304_21_PCRleft
TTGACACTAATCTCTGCTTGTGTTCTCU


 


95
000038_00_PCRright
AAUGGAUAAACUACAATUAAAAGUCACAGUCU


 


96
000038_01_PCRright
CACCCAAAUCGAGAGAAGCUGUACU


 


97
000038_02_PCRright
CACAAGGCAAUGUTUACUAUAUGAAGAAAAGU


 


98
000038_03_PCRright
AAAGUTUCAAAUAAGTUGUACUGCCAAGU


 


99
000038_04_PCRright
TUCGCUGUTTUAUCACTUAGAAACAAGU


 


100
000038_05_PCRright
UACCCACAAACAAGAAAGGCAAUTU


 


101
000038_06_PCRright
GACAGCACATUGGUACUGAAUGCTU


 


102
000038_07_PCRright
CCCAAAAUGCUGGGATUACAGGU


 


103
000038_08_PCRright
UTUCUGUTUAAAAAUTUCACAUTUGCTU


 


104
000038_09_PCRright
CAGAGGAAGCAGCUGAUAACAGAAGU


 


105
000038_10_PCRright
GCGAAUGUGAAGCACAGGUTTUUAU


 


106
000038_11_PCRright
GGCUGAAGUGGGAGGATUGCU


 


107
000038_12_PCRright
UGAAUAAUACACAGGUAAGAAATUAGGAAAUCU


 


108
000038_13_PCRright
GCTUAAAACUTUCAUGATUAUAUAAAACATUGCU


 


109
000249_00_PCRright
GCAUGCGCUGUACAUGCCUCU


 


110
000249_01_PCRright
GCCUAGUTUCCAGAACAGAGAAAGGU


 


111
000249_02_PCRright
GGAGGAUAUTTUACACAUTUCTUGAAUCUTU


 


112
000249_03_PCRright
CACUGGUGTUGAGACAGGATUACUCU


 


113
000249_04_PCRright
GCTUCAACAAUTUACUCUCCCAUGU


 


114
000249_05_PCRright
UCUCAGAGACCCACUCCCAGAU


 


115
000249_06_PCRright
GGCUGAGACUGAAACAUCAUAACCTU


 


116
000249_07_PCRright
CAAAUCUGAAGCAUAAAACAAGCCU


 


117
000249_08_PCRright
UTUCCAUGGUCCCAUAAAATUCCCU


 


118
000249_09_PCRright
CUGUAAGAAGGGACAGAACAUCCTU


 


119
000249_10_PCRright
AAUAACAGGCAAAAAUCUGGGCUCU


 


120
000249_11_PCRright
GCUGUACUTTUCCCAAAAGGCCAU


 


121
000249_12_PCRright
AAACCTUGGCAGTUGAGGCCCUAU


 


122
000249_13_PCRright
GGAUTUGAAACCACAUGUGUCUGACU


 


123
000249_14_PCRright
GAAAUTUCAGAAGUGAAAAGGAUCUAAACU


 


124
000249_15_PCRright
ACCCCAAGTUAUCUGCCCACCU


 


125
000249_16_PCRright
AAAGGGUGGUCAUTUGCCCUTU


 


126
000249_17_PCRright
TUGUAUGAGGUCCUGUCCUAGUCCU


 


127
000249_18_PCRright
UCGGAAUACAGAGAAAGAAGAACACAU


 


128
000321_00_PCRright
ACGGCGGCUCUGCUCGCU


 


129
000321_01_PCRright
TUCAAUTTUUGUAUAGUGAUTUGAAGTUGTU


 


130
000321_02_PCRright
TUGAGAGGAAAAUCCAGAATUCGTU


 


131
000321_03_PCRright
UGAGCUAACATUAAAAGGGACAAGUCU


 


132
000321_04_PCRright
UCUACACAGGACTUAAAUCUAUGGGCTU


 


133
000321_05_PCRright
GCAGAGAAUGAGGGAGGAGUACATU


 


134
000321_06_PCRright
AUCAUCCUGUCAGCCTUAGAACCAU


 


135
000321_07_PCRright
AAAAACAUGCUCAUAACAAAAGAAGUAAAU


 


136
000321_08_PCRright
GACAATUAUCCUCCCUCCACAGUCU


 


137
000321_09_PCRright
CCUAUAUCUAAAGCAAAUCAAUCAAAUAUACCAU


 


138
000321_10_PCRright
UGAAUACAUAAAGAAACGUGAACAAAUCU


 


139
000321_11_PCRright
UCAAGUTUCUTUGCCAAGAUATUACAAUAAAUAAU


 


140
000321_12_PCRright
CGAACUGGAAAGAUGCUGCUTTUAAU


 


141
000321_14_PCRright
AGCGCACGCCAAUAAAGACAU


 


142
000321_15_PCRright
GCATUCCTUCUCCTUAACCUCACACU


 


143
000321_16_PCRright
AGAUGTUAAGAAACACCUCUCACUAACAAU


 


144
000321_17_PCRright
UGCAGUTUGAAUGGUCAACAUAACAU


 


145
000321_18_PCRright
AACAUGAUTUGAACCCAGUCAGCCU


 


146
000321_19_PCRright
GAGGAGAGAAGGUGAAGUGCTUGAU


 


147
000321_20_PCRright
UGAATUACCUAUGTUAUGTUAUGGAUAUGGAUTUAU


 


148
000321_21_PCRright
AAGGGCTUCGAGGAAUGUGAGGUAU


 


149
000321_22_PCRright
UCAAAAUAAUCCCCCUCUCATUCUTU


 


150
000321_23_PCRright
UAUGCAAUAUGCCUGGAUGAGGUGU


 


151
000321_24_PCRright
AACTUGGCAUGAAAGAAATUGGUAU


 


152
000321_25_PCRright
AAACAAACCUGCCAACUGAAGAAAU


 


153
000321_26_PCRright
UGUGAGAGACAAUGAAUCCAGAGGU


 


154
000546_00_PCRright
ACAGGUCUCUGCUAGGGGGCU


 


155
000546_01_PCRright
GACAGCAUCAAAUCAUCCATUGCU


 


156
000546_02_PCRright
UCCCAAAGTUCCAAACAAAAGAAAU


 


157
000546_03_PCRright
GCAAAUTUCCTUCCACUCGGAU


 


158
000546_04_PCRright
CUCCUCCCAGAGACCCCAGTU


 


159
000546_05_PCRright
GGUCAGAGGCAAGCAGAGGCU


 


160
000546_06_PCRright
GAAUCUGAGGCAUAACUGCACCCU


 


161
000546_07_PCRright
AGCUACAACCAGGAGCCATUGUCTU


 


162
000546_08_PCRright
CAACCUAGGAAGGCAGGGGAGU


 


163
000546_09_PCRright
CGGGACAAAGCAAAUGGAAGU


 


164
000551_00_PCRright
CTUCAGACCGUGCUAUCGUCCCU


 


165
000551_01_PCRright
AAAGATUGGAUAACGUGCCUGACAU


 


166
000551_02_PCRright
GAAACUAAGGAAGGAACCAGUCCUGU


 


167
007304_00_PCRright
CCCAAATUAAUACACUCTUGUGCUGACU


 


168
007304_01_PCRright
UGGAGCCACAUAACACATUCAAACU


 


169
007304_02_PCRright
TUCUACUTTUUCCUACUGUGGTUGCTU


 


170
007304_03_PCRright
AGCACTUGAGUGUCATUCTUGGGAU


 


171
007304_04_PCRright
GGCUAAGGCAGGAGGACUGCTU


 


172
007304_05_PCRright
UCACCAUAGGGCUCAUAAAATUCACU


 


173
007304_06_PCRright
GGAAAAUACCAGCTUCAUAGACAAAGGU


 


174
007304_07_PCRright
AACUCUGCCAAGAGAUTTUGUGGGU


 


175
007304_08_PCRright
GCUGUAAUGAGCUGGCAUGAGUAUTU


 


176
007304_09_PCRright
TUGUGCCATUAATUCAAAGAGAUGAU


 


177
007304_10_PCRright
AAGGCUCCAUAATUACCCAUGUGCU


 


178
007304_11_PCRright
CCACAGCAUCUTUACATUGAUGUTUCU


 


179
007304_12_PCRright
UGUTUGTUCCAAUACAGCAGAUGAAAU


 


180
007304_13_PCRright
UGTUGTUAAGUCTUAGUCATUAGGGAGAUACAU


 


181
007304_14_PCRright
CAAAGUGCUGCGATUACAGGCAU


 


182
007304_15_PCRright
GGUGUAAAAAUGCAATUCUGAGGUGTU


 


183
007304_16_PCRright
UTUGUGCATUGTUAAGGAAAGUGGU


 


184
007304_17_PCRright
GGUGGGGUGAGAUTTUUGUCAACTU


 


185
007304_18_PCRright
UCCACUAUGUAAGACAAAGGCUGGU


 


186
007304_19_PCRright
GAGGCUACAGUAGGGGCAUCCAU


 


187
007304_20_PCRright
CAAAAGGACCCCAUAUAGCACAGGU


 


188
007304_21_PCRright
GGGGUCCUGUGGCUCUGUACCU
















TABLE B







Nested Patch











SEQ





ID.





NO.
Oligo Name
Sequence















189
000038_00_PP L
TTAGTGGCTGCTTGTTTTTAAAGAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



190
000038_00_PP R
CAAGCAGAAGACGGCATACGATGATACCTTCATATTAGATGCCTCAGT



 



191
000038_01_PP L
TTTCTTGACATTTAAGTATGCTGAGAAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



192
000038_01_PP R
CAAGCAGAAGACGGCATACGATGGATCTACACACCTAAAGATGACA



 



193
000038_02_PP L
GCTTTAAGCAGTCTAAAATATTCTTAATGTTATATTATTTTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



194
000038_02_PP R
CAAGCAGAAGACGGCATACGATACCTCTCTTTCTCAAGTTCTTCTAAATATC



 



195
000038_03_PP L
AAGACTGCAGAAGAGCAATACTTACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



196
000038_03_PP R
CAAGCAGAAGACGGCATACGATACTTACATTTTCAGTTAAAGGAAGACTATCT



 



197
000038_04_PP L
CCAATAAAGAAAATGAATAAGCAAATACGTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



198
000038_04_PP R
CAAGCAGAAGACGGCATACGAAACTTACCTGTGCTCGTTTTTCCAT



 



199
000038_05_PP L
TACTATGGCTACCACTTAAAAGCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



200
000038_05_PP R
CAAGCAGAAGACGGCATACGAACTAACCTCTGCTTCTGTTGCTTG



 



201
000038_06_PP L
ACATCAGTACATGCAAAAATGGTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



202
000038_06_PP R
CAAGCAGAAGACGGCATACGACTGGAAATATGCATTCAGGACTAAGA



 



203
000038_07_PP L
ACTCCAAATGAAGTGTCTGTATGATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



204
000038_07_PP R
CAAGCAGAAGACGGCATACGAGTGAGCCACTGCACCTGG



 



205
000038_08_PP L
CACCTGTGGGCCAAATGAGTTTAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



206
000038_08_PP R
CAAGCAGAAGACGGCATACGATGAAACATGCACTACGATGTACACT



 



207
000038_09_PP L
GCAGGGATCACTAATATAACCCTAATTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



208
000038_09_PP R
CAAGCAGAAGACGGCATACGATGGTGGCCTTATATCCTAATTCATC



 



209
000038_10_PP L
TGGCCTGTAGTCCCCCTAATTTAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



210
000038_10_PP R
CAAGCAGAAGACGGCATACGACAGTCATTGTTTAATGAGGAGAGTGA



 



211
000038_11_PP L
GCCTGTAAATTAAATACAGAATAGAGGATCATTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



212
000038_11_PP R
CAAGCAGAAGACGGCATACGATGAACCCTGGAGGCAGAGG



 



213
000038_12_PP L
GAAATTCTGGCTAGCCGTGGTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



214
000038_12_PP R
CAAGCAGAAGACGGCATACGACATGGCTAAAAGAAGGCAGCAAAAA



 



215
000038_13_PP L
AGTAAGAAACAGAATATGGGTCATCTAATTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



216
000038_13_PP R
CAAGCAGAAGACGGCATACGATACAATTAGGTCTTTTTGAGAGTATGAATTC



 



217
000249_00_PP L
TGGCGCCAGAAGAGCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



218
000249_00_PP R
CAAGCAGAAGACGGCATACGAGCCCGGGCAAAGAGGC



 



219
000249_01_PP L
CTCCAAATACAAACAATAGTGCCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



220
000249_01_PP R
CAAGCAGAAGACGGCATACGACCTGACTCTTCCATGAAGCGC



 



221
000249_02_PP L
ATGTTACTCATTTTTCCAAATCTCTTTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



222
000249_02_PP R
CAAGCAGAAGACGGCATACGAAGCTTACCTCACCTCGAAAGCC



 



223
000249_03_PP L
TCACCCACTGTCACCTCACCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



224
000249_03_PP R
CAAGCAGAAGACGGCATACGAGAGACCTAGGCAAAAAATACATTTCAG



 



225
000249_04_PP L
ATCCAGTAGAGAGATAGATACTAATCCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



226
000249_04_PP R
CAAGCAGAAGACGGCATACGAACCATTCTTACCGTGATCTGGGTC



 



227
000249_05_PP L
AAATAAAACCCAAGATGTCCTGGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



228
000249_05_PP R
CAAGCAGAAGACGGCATACGATTTGGACTGTACCTGCCAACAACT



 



229
000249_06_PP L
CAAAAGAGTAAGAAAAGAGTTGCCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



230
000249_06_PP R
CAAGCAGAAGACGGCATACGAATCTCCACCAGCAAACTATTAAAAATC



 



231
000249_07_PP L
CAGCTACTGTCTCTCCTTGCTGATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



232
000249_07_PP R
CAAGCAGAAGACGGCATACGAGTGTATTTGACTAAAGCAAACTCTTAACA



 



233
000249_08_PP L
TTTGTGAAATGAGGGCCCCGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



234
000249_08_PP R
CAAGCAGAAGACGGCATACGAGTGGGTGTTTCCTGTGAGTGGAT



 



235
000249_09_PP L
GGGGTGAGGTCACAGGTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



236
000249_09_PP R
CAAGCAGAAGACGGCATACGATTGCCAGTGGTGTATGGGATTCA



 



237
000249_10_PP L
AGGGGGAGAAAAAGCCCACATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



238
000249_10_PP R
CAAGCAGAAGACGGCATACGACACGTCTGGCCGGGC



 



239
000249_11_PP L
AGTGGAGAGACTCAGAATAAGAAGTATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



240
000249_11_PP R
CAAGCAGAAGACGGCATACGAACCTGGGGTTGCTGGAAGTAGG



 



241
000249_12_PP L
GTTGCATTTTGGAGGAGCAAGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



242
000249_12_PP R
CAAGCAGAAGACGGCATACGAGCATCCCAGGCAGGCC



 



243
000249_13_PP L
AAGCACCAGGCACCAGAACTAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



244
000249_13_PP R
CAAGCAGAAGACGGCATACGACCAAAGCCTGTGCCCTCC



 



245
000249_14_PP L
AACCAGTTGGGACAAAATGGGAGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



246
000249_14_PP R
CAAGCAGAAGACGGCATACGATACCGATAACCTGAGAACACCAAAA



 



247
000249_15_PP L
CGGTGCTGGCTCCTAGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



248
000249_15_PP R
CAAGCAGAAGACGGCATACGACAGCCTCCCAAAGTGCTGG



 



249
000249_16_PP L
GCCTTGTGCTCCTATCTGCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



250
000249_16_PP R
CAAGCAGAAGACGGCATACGACCCTCCAGCACACATGCATG



 



251
000249_17_PP L
TGTGATACTTTAGGCGTTAAAACTGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



252
000249_17_PP R
CAAGCAGAAGACGGCATACGAGGGGTGCCAGTGTGCATC



 



253
000249_18_PP L
GCCTCCCTGTTTGCATCCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



254
000249_18_PP R
CAAGCAGAAGACGGCATACGACCCACAGTGCATAAATAACCATATTT



 



255
000321_00_PP L
AACTGAGCGCCGCGTCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



256
000321_00_PP R
CAAGCAGAAGACGGCATACGACACCTGACGAGAGGCAGGTC



 



257
000321_01_PP L
TGTTTCAATAGTTTGCACATAACACTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



258
000321_01_PP R
CAAGCAGAAGACGGCATACGATTTAAAATGAGAAAAAAAAATTTCAAAACGTTTTAAG



 



259
000321_02_PP L
TTTCTTATTCAGCATACAAAATAAATGTTTGTAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



260
000321_02_PP R
CAAGCAGAAGACGGCATACGATCCTTTTATGGCAGAGGCTTATATT



 



261
000321_03_PP L
TTCAATTCAAAAGATTATCAGCTCTACATCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



262
000321_03_PP R
CAAGCAGAAGACGGCATACGAAAGAATTAATACTTACTAACTTTACTAAATGTGTTAAATAATT



 



263
000321_04_PP L
TTTTTAACATTTTTTCGTAATTTAGAAGTCATAGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



264
000321_04_PP R
CAAGCAGAAGACGGCATACGAAATTTATGAAGTAGCCTGCTATAATCGA



 



265
000321_05_PP L
TGTATCACTGAAAGAAAGTTTTCCAGATATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



266
000321_05_PP R
CAAGCAGAAGACGGCATACGAACTCAATAAAAATTGGGGAATTTAGTCC



 



267
000321_06_PP L
CGCAGGGTAGAGTATATCCATAAATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



268
000321_06_PP R
CAAGCAGAAGACGGCATACGAGTTTGGTACCCACTAGACATTCAAT



 



269
000321_07_PP L
ATGGGTATAACAGCTGTTTCTGTAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



270
000321_07_PP R
CAAGCAGAAGACGGCATACGAATTGTTAGGGAGAACTTACATCTAAATCT



 



271
000321_08_PP L
CTTGACTCTTGAACAATGCAGGGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



272
000321_08_PP R
CAAGCAGAAGACGGCATACGACAAAACATTAATATTTTATTAAATTTCCTTTCAGATTACC



 



273
000321_09_PP L
CATGTCATTACATCTCTCAGCACACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



274
000321_09_PP R
CAAGCAGAAGACGGCATACGAGTGCAATACCTGTCTATAGAATCAGT



 



275
000321_10_PP L
TGCTTTATGCATCAAAAAAGCAGTATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



276
000321_10_PP R
CAAGCAGAAGACGGCATACGAGAAACACTATAAAGCCATGAATAACAAAATT



 



277
000321_11_PP L
CACTGCCTCCCACTTGTCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



278
000321_11_PP R
CAAGCAGAAGACGGCATACGAGTTTCATATATGGCTTACGTTAAAATAGGA



 



279
000321_12_PP L
TTTTGGATTCACTGTGCAGTTCTTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



280
000321_12_PP R
CAAGCAGAAGACGGCATACGAATTATTACTCTATAGTACCACGAATTACAATGA



 



281
000321_14_PP L
TTGCCAGGCTGGGGTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



282
000321_14_PP R
CAAGCAGAAGACGGCATACGAATGAAAAATGTTGTCATTCAGAAGTTTGC



 



283
000321_15_PP L
ACTAAAAGTAAAAAATTTACCTAAAATTTTGAATGGATAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



284
000321_15_PP R
CAAGCAGAAGACGGCATACGAATCCCTCTCCCCCGACCA



 



285
000321_16_PP L
TGAGCTAGGTATTTTTTTGGAAGTTATTATCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



286
000321_16_PP R
CAAGCAGAAGACGGCATACGAAATTTGTTAGCCATATGCACATGAA



 



287
000321_17_PP L
AGTACTATGAATTTTAGGCACAATTGACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



288
000321_17_PP R
CAAGCAGAAGACGGCATACGAATATTTTGCTTACATATCTGCTGCAG



 



289
000321_18_PP L
CAAGTTGGCTAAGAATCACAGATTATACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



290
000321_18_PP R
CAAGCAGAAGACGGCATACGAAGTTTCAGAGTCCATGCTCTTGAAA



 



291
000321_19_PP L
GTAGCATTTTAACAGAAACCTCTTTTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



292
000321_19_PP R
CAAGCAGAAGACGGCATACGATTTCTTACTTGGTCCAAATGCCTGT



 



293
000321_20_PP L
AATACCATTTTCTTTCTTTTAGCCTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



294
000321_20_PP R
CAAGCAGAAGACGGCATACGACAAAAAAACTTACTATGGAAAATTACCTACCT



 



295
000321_21_PP L
ACCTTTAGATTTTCTTTTCTAATAGTTTATAATACTTTTTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



296
000321_21_PP R
CAAGCAGAAGACGGCATACGATGGTGACAAGGTAGGGGGC



 



297
000321_22_PP L
CCTGGTGGAAGCATACTGCAAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



298
000321_22_PP R
CAAGCAGAAGACGGCATACGAACTACTTCCCTAAAGAGAAAACACAC



 



299
000321_23_PP L
ACAATTTTGCAGAGATGAGCATAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



300
000321_23_PP R
CAAGCAGAAGACGGCATACGATTGAATAACTGCATTTGGAAATTCAAATTAT



 



301
000321_24_PP L
CATAGTTAGCAACCTCAAGTTATAGTTTGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



302
000321_24_PP R
CAAGCAGAAGACGGCATACGAAAGCCAGGAGCAGTGCTGA



 



303
000321_25_PP L
TGGAAAACTCAAATTTCCAGTAACTATGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



304
000321_25_PP R
CAAGCAGAAGACGGCATACGATATACATTCTTTTATATAACGAAAAGACTTCTTGC



 



305
000321_26_PP L
GCGCTCAGGACCTTGCAAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



306
000321_26_PP R
CAAGCAGAAGACGGCATACGAGTACACAGTGTCCACCAAGGTC



 



307
000546_00_PP L
GGAACCCCCTCCCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



308
000546_00_PP R
CAAGCAGAAGACGGCATACGAGGGGTTGGGGTGGGG



 



309
000546_01_PP L
CCTGCCCTTCCAATGGATCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



310
000546_01_PP R
CAAGCAGAAGACGGCATACGATGGGACGGCAAGGGGG



 



311
000546_02_PP L
CCAGGTCCCCAGCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



312
000546_02_PP R
CAAGCAGAAGACGGCATACGAGCAGGGGGATACGGCCA



 



313
000546_03_PP L
CAACTGGAAGACGGCAGCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



314
000546_03_PP R
CAAGCAGAAGACGGCATACGAAAGATGCTGAGGAGGGGCC



 



315
000546_04_PP L
GGGGCAGCGCCTCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



316
000546_04_PP R
CAAGCAGAAGACGGCATACGAGCAAACCAGACCTCAGGCG



 



317
000546_05_PP L
GCCCAGGCTGGAGTGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



318
000546_05_PP R
CAAGCAGAAGACGGCATACGAGGGGCACAGCAGGCC



 



319
000546_06_PP L
ACCAGGCTCCATCTACTCCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



320
000546_06_PP R
CAAGCAGAAGACGGCATACGATGGTCTCCTCCACCGCTTC



 



321
000546_07_PP L
GGTGTTGTTGGGCAGTGCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



322
000546_07_PP R
CAAGCAGAAGACGGCATACGATGAGGCATCACTGCCCCC



 



323
000546_08_PP L
GCCCACGGATCTGCAGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



324
000546_08_PP R
CAAGCAGAAGACGGCATACGAAGGGCCAGGAAGGGGC



 



325
000546_09_PP L
GGGCCTAAGGCTGGGACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



326
000546_09_PP R
CAAGCAGAAGACGGCATACGACCTGGGTGCTTCTGACGC



 



327
000551_00_PP L
TCTTCGCGCGCGCTCGGTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



328
000551_00_PP R
CAAGCAGAAGACGGCATACGAGCTGGGTCGGGCCTAAG



 



329
000551_01_PP L
GGCACGGTGGCCCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



330
000551_01_PP R
CAAGCAGAAGACGGCATACGACAGGCAAAAATTGAGAACTGGGCTT



 



331
000551_02_PP L
CTCAGTGGCAGACTAGGGTCTCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



332
000551_02_PP R
CAAGCAGAAGACGGCATACGAATCTAGATCAAGACTCATCAGTACCA



 



333
007304_00_PP L
ATGACAACTTCATTTTATCATTTTAAAATAAAGTAAATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



334
007304_00_PP R
CAAGCAGAAGACGGCATACGATACCAGATGGGACACTCTAAGATTT



 



335
007304_01_PP L
CTAGCAGGGTAGGGGGGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



336
007304_01_PP R
CAAGCAGAAGACGGCATACGATACTTGCAAAATATGTGGTCACACT



 



337
007304_02_PP L
TCAAAAGGCAAATAGCCATGAAAAGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



338
007304_02_PP R
CAAGCAGAAGACGGCATACGACCAACCTAGCATCATTACCAAATTATATAC



 



339
007304_03_PP L
TACTTTCTTGTAGGCTCCTGAAATTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



340
007304_03_PP R
CAAGCAGAAGACGGCATACGAATTCAACACTTACACTCCAAACCTG



 



341
007304_04_PP L
CCCTATGTATGCTCTTTGTTGTGTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



342
007304_04_PP R
CAAGCAGAAGACGGCATACGACTAGCCTGGGCCACAGAG



 



343
007304_05_PP L
AAGAACAGTCAAGCAATTGTTGGCCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



344
007304_05_PP R
CAAGCAGAAGACGGCATACGATCCCAAAGCTGCCTACCACAAATA



 



345
007304_06_PP L
AGATATTCAACTAGAAATATTTACTGAGCATCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



346
007304_06_PP R
CAAGCAGAAGACGGCATACGATCTCTTTGACTCACCTGCAATAAGT



 



347
007304_07_PP L
GATTACAGAAAGCTGACCAATCTTATTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



348
007304_07_PP R
CAAGCAGAAGACGGCATACGATGTAAAGGTCCCAAATGGTCTTCAG



 



349
007304_08_PP L
TCACAAGCAGCTGAAAATATACAAAAATGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



350
007304_08_PP R
CAAGCAGAAGACGGCATACGAGTGCCACATGGCTCCACATG



 



351
007304_09_PP L
AGGACTGGATTTACTTTCATGTCACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



352
007304_09_PP R
CAAGCAGAAGACGGCATACGAGTCAGCAAACCTAAGAATGTGGGAT



 



353
007304_10_PP L
TTGCATGGTATCCCTCTGCTTCAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



354
007304_10_PP R
CAAGCAGAAGACGGCATACGAGAGCAAGGATCATAAAATGTTGGAG



 



355
007304_11_PP L
ACTGCTTTAAATGGAATGAGAAAACAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



356
007304_11_PP R
CAAGCAGAAGACGGCATACGATACCTTTCCACTCCTGGTTCTTTAT



 



357
007304_12_PP L
GCTGGGCAGCCAAAGCATAAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



358
007304_12_PP R
CAAGCAGAAGACGGCATACGAATTACCTAGATCTTGCCTTGGCAAG



 



359
007304_13_PP L
CAGGTAAGGGGTTCCCTCTGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



360
007304_13_PP R
CAAGCAGAAGACGGCATACGAATGGATACACTCACAAATTCTTCTGG



 



361
007304_14_PP L
ATTCCACCATGGCATATGTTTACCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



362
007304_14_PP R
CAAGCAGAAGACGGCATACGAGCGCCACCGTGCCTC



 



363
007304_15_PP L
AGAAGCTAAAGAGCCTCAGTTTTTTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



364
007304_15_PP R
CAAGCAGAAGACGGCATACGAAAAGGGAGGAGGGGAGAAATAGTAT



 



365
007304_16_PP L
CAGAGGAGAGGTCCTTCCCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



366
007304_16_PP R
CAAGCAGAAGACGGCATACGAGCATTGATGGAAGGAAGCAAATACA



 



367
007304_17_PP L
CATTCAGGCCAGGCGCGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



368
007304_17_PP R
CAAGCAGAAGACGGCATACGAGAGGGAGGGAGCTTTACCTTTCTG



 



369
007304_18_PP L
TGGAAGAAGAGAGGAAGAGAGAGGGGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



370
007304_18_PP R
CAAGCAGAAGACGGCATACGAGCTGGAACTCTGGGGTTCTCC



 



371
007304_19_PP L
GCATACTTAACCCAGGCCCTCTGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



372
007304_19_PP R
CAAGCAGAAGACGGCATACGAAGGGACTGACAGGTGCCAG



 



373
007304_20_PP L
CCTGGATCCCCAGGAAGGAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



374
007304_20_PP R
CAAGCAGAAGACGGCATACGAACATGCAGGCACCTTACCATG



 



375
007304_21_PP L
CATCTGCCCAATTGCTGGAGACGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT



 



376
007304_21_PP R
CAAGCAGAAGACGGCATACGAGTGGCTGGCTGCAGTCAG


















TABLE C







SEQ ID.










NO.
Oligo Name
Sequence





377
Upstream Universal Primer For Ligation
ACACTCTTTCCCTACACGACGCTCTTCCGATC


 


378
Downstream Universal Primer For
5′ Phosphate TCGTATGCCGTCTTCTGCTTG 3′



Ligation



 


379
Final Universal PCR Barcode Forward
GCCTCCCTCGCGCCATCAGCTACACGACGCTCTTCCGATC



Primer for Normal Sample



 


380
Final Universal PCR Barcode Reverse
GCCTTGCCAGCCCGCTCAGCAAGCAGAAGACGGCATACGA



Primer for Normal Sample



 


381
Final Universal PCR Barcode Forward
GCCTCCCTCGCGCCATCAGGTCACACTACACGACGCTCTTCCGATC



Primer for Colon Cancer Sample



 


382
Final Universal PCR Barcode Reverse
GCCTTGCCAGCCCGCTCAGCAGTCACAAGCAGAAGACGGCATACGA



Primer for Colon Cancer Sample









Nucleic Acid Patch PCR

Genomic DNA from a moderately differentiated colon adenocarcinoma primary tumor and adjacent normal tissue from an 81-year-old male (Biochain catalog # D8235090-PP-10) was used as template for the first PCR. Targets were amplified in a reaction containing 1 μg human genomic DNA, 50 nM each of 94 Forward PCR primers, 50 nM each of 94 Reverse PCR primers, 5 units of AmpliTaq Polymerase Stoffel Fragment (Applied Biosystems), 200 μM each dNTP, 2 mM MgCl2, 20 mM Tris-HCl pH 8.4 and 50 mM KCl in a total volume of 10 μl. This reaction was incubated at 94° C. for 2 min followed by (94° C. for 30 sec, 56° C. for 30 sec, 72° C. for 6 min)×10 cycles, and then held at 4° C.
To prepare for the next round of oligonucleotide hybridization, the uracil-containing primers from the first reaction were cleaved from the amplicons by the addition of 1 unit heat labile Uracil-DNA Glycosylase (USB), 10 units of Endonuclease VIII (NEB), and 10 units of Exonulcease I (USB). This mix was incubated at 37° C. for 2 hours followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C. To remove the unincorporated nucleotide from the mix, 0.05 U Apyrase (NEB) was added to the reaction and incubated at 30° C. for 30 minutes.
Nucleic acid patch-driven ligation of the universal primers to correct amplicons is performed by addition of more reactants to the initial tube to result in the following final concentrations: 20 nM each nucleic acid patch oligonucleotide, 40 nM Universal Primer 1, 40 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 μl. This reaction was incubated at 95° C. for 15 min followed by (94° C. for 30 sec, 65° C. for 2 min, 55° C. for 1 min, 60° C. for 5 min) for 100 cycles, and held at 4° C.
Incorrect products, template genomic DNA and excess primer were degraded by the addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre). This mix was incubated at 37° C. for 2 hours followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C. Each selection reaction was purified using a Qiaquick Spin Column (Qiagen) and the final elution was performed with 30 μl elution buffer (EB).
For the final PCR using the universal primers, reagents were added to the elution to result in these final concentrations in 50 μl: 0.5 μM each Tailed Universal Primer (see below), 10 U Platinum Taq Polymerase (Invitrogen) 0.5 mM each dNTP, 2 mM MgCl2, 0.5 M Betaine to improve the amplification of GC-rich sequences, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 min followed by (93° C. for 30 sec, 60° C. for 6 min) for 27 cycles, and held at 4° C. The universal PCR used the Final Universal PCR primers tailed with 454 Life Sciences A or B oligonucleotide at the 5′ end, followed by a sample-specific 6 bp sequence and ending at the 3′ end with the same universal primer sequence ligated to the amplicons in the nucleic acid patch PCR procedure. The PCR product smear between the expected sizes was confirmed by running on a 3% Metaphor Agarose gel (Lonza). The reactions were then purified on a Qiaquick Spin Column (Qiagen). The eluted DNA was quantified on a Nanodrop spectrophotometer (ThermoFisher Scientific Inc.), and the same quantity of DNA was pooled together from the two separate samples. This pooled sample was sequenced using the 454 sequencing system on the 454 Life Sciences/Roche FLX machine.
Sequence Analysis
To determine which sequences matched the intended targets, the reads were aligned against a database of reference target sequences for each target using the BLASTN software at the Washington university in St. Louis BLAST archives (http://blast.wustl.edu). The number of reads that matched significantly to each exon was determined (p<0.02). The first six bases of sequence from each read, the sample specific DNA barcode, was used to determine whether the sequence came from the tumor sample or the normal sample. The number of reads that did not match targeted sequence was determined, and those sequences were aligned to a database of nucleic acid patch oligonucleotide sequence to identify what fraction was due to primer artifacts. For each exon, CLUSTALW was used to generate a multiple sequence alignment of all of the reads against the reference sequence (Larkin, Blackshields, Brown, Chenna, McGettigan, McWilliam, Valentin, Wallace, Wilm, Lopez et al. 2007). The majority of the differences from the reference sequence were insertion or deletion mutations (indels) adjacent to stretches of identical nucleotides (homopolymers), which is a known error-prone feature for 454 sequencing (Ronaghi, Uhlen and Nyren 1998). To filter these out, all the positions that did not match the reference sequence but were in greater than 30% of the reads were examined.
Results
Oligonucleotides were designed for 94 of the 96 exons from the six nucleotide sequences encoding colon-cancer related proteins. Attempts to design oligonucleotides to two of the 96 exons failed; the last exon of APC failed because of length ( ̃6000 bp) and an exon in RB1 failed due to the presence of Alu repeat elements surrounding the exon.
55,068 sequencing reads were obtained. At least one read from each sample was mapped to 90 of the 94 exons (95.7%). The 4 exons that failed to amplify were due to imperfect primer/patch design. Two of the loci could not be amplified in separate individual PCR reactions, indicating PCR primer failure. The other 2 loci failed because their patch oligonucleotides bound to multiple locations in the genome. This problem could be avoided by more careful primer design. Ninety percent of all reads (49553 reads) mapped to one of the targeted exons. Thus, a 125,000-fold enrichment was achieved with nucleic acid patch PCR from genomic DNA (90% specificity×total possible fold enrichment). When selecting a fraction of the genome this small, the total possible enrichment is 138,888 fold (3×109 bp genome/21.6 kbp targeted). Of the remaining 10% of reads that did not match the targeted regions, most (85%) appear to be due to concatamers of nucleic acid patch oligonucleotides that contain Alu elements. It is likely that designing oligonucleotides that do not overlap repetitive genomic elements could reduce this background.
These results demonstrate that nucleic acid patch PCR can be performed on multiple samples in parallel, which can then be labeled with sample-specific DNA barcodes and sequenced as a pool. The choice of targets and target boundaries is flexible, and a wide range of sizes can be amplified simultaneously (here, 74 bp to 438 bp). Nucleic acid patch PCR is robust and sensitive, as this method was able to amplify 90 of the 94 targeted exons.
Example 3: Uniformity of Nucleic Acid Patch PCR Per Exon in Each Sample
Ideally for any multiplexed PCR method, all targeted regions would be uniformly amplified within each reaction by all primer pairs, and across samples from different templates. To analyze the uniformity of amplification of the 90 regions generated by nucleic acid patch PCR in Example 2, the number of reads obtained for each targeted was graphed (FIG. 3A). The number of sequencing reads obtained for each exon is also presented numerically in TABLE D. Sequence coverage ranged over 2-3 logs (base 10), with 75% (68/90) of exons having between 10 and 500 reads in both samples (50 fold abundance range). The median number of reads per exon was 145. Seventy-six percent of all exons fell within 5-fold coverage of this median (29-725 reads). There were no parameters found that explain the non-uniformity. Exon non-uniformity did not correlate with the gene, the size of the amplicon, nor the GC content of the oligonucleotides.







TABLE D







Reads Per Exon in Tumor and Normal Samples










Number of Reads
Number of Reads


RefSeq _ Exon Number
in Normal Sample
in Tumor Sample












NM_000551_1
1
2


NM_000321_22
3
8


NM_000546_5
4
12


NM_007304_0
7
3


NM_007304_2
7
8


NM_000546_3
10
5


NM_000546_9
10
15


NM_000321_16
13
9


NM_007304_4
15
24


NM_000038_8
16
31


NM_000321_20
16
26


NM_000249_8
17
17


NM_000321_9
28
30


NM_000321_26
32
14


NM_000321_17
33
38


NM_007304_10
33
33


NM_000321_14
38
98


NM_000321_24
42
65


NM_000321_1
44
129


NM_000038_2
47
55


NM_000551_2
47
113


NM_000321_7
48
17


NM_000038_7
56
59


NM_000321_2
60
83


NM_000249_17
63
43


NM_000546_8
63
59


NM_007304_21
66
115


NM_000038_11
68
34


NM_000321_4
72
65


NM_000321_5
75
89


NM_000321_6
79
100


NM_000321_10
80
65


NM_000038_12
83
75


NM_000321_18
87
98


NM_007304_7
88
58


NM_007304_9
95
98


NM_007304_14
96
87


NM_007304_16
105
118


NM_000546_0
108
85


NM_000249_1
111
150


NM_000038_13
121
70


NM_007304_8
124
114


NM_000321_11
129
97


NM_000038_5
133
71


NM_000249_12
143
91


NM_000546_1
148
92


NM_007304_15
149
109


NM_000249_7
154
149


NM_007304_20
159
178


NM_007304_19
160
134


NM_007304_13
162
160


NM_007304_11
165
74


NM_000249_10
200
136


NM_000321_3
222
243


NM_000249_0
235
146


NM_007304_17
247
227


NM_000321_15
253
265


NM_000249_15
267
145


NM_000546_6
283
151


NM_007304_12
286
270


NM_000249_2
288
249


NM_000249_9
292
225


NM_000038_4
314
227


NM_000321_19
317
464


NM_000038_3
332
284


NM_000249_13
353
199


NM_000038_9
356
316


NM_000321_25
386
301


NM_000546_7
396
200


NM_000546_4
415
206


NM_000321_21
416
373


NM_007304_5
464
358


NM_000249_18
498
340


NM_000038_10
524
338


NM_007304_18
532
373


NM_000038_0
549
273


NM_000249_6
587
470


NM_000249_3
648
433


NM_000249_4
660
574


NM_000321_12
670
434


NM_000038_1
713
320


NM_007304_3
940
833


NM_000249_14
942
483


NM_000249_16
948
471


NM_007304_6
975
779


NM_000038_6
1170
780


NM_000321_23
1198
967


NM_000321_8
1283
697


NM_007304_1
1932
1605


NM_000249_5
2813
1665









To test the reproducibility of the nucleic acid patch PCR method, the number of reads per exon from the tumor and normal samples were correlated. The correlation was high (R2 of 93%), indicating high reproducibility (FIG. 4A). In fact, 85% (77/90) of exons displayed at most a 2 fold difference in abundance between samples, and all exons were within 3 fold relative abundance between samples (FIG. 4B).
These results demonstrate that even though the abundance of PCR products varies between exons, the abundance of each exon is highly reproducible across different reactions and samples.
Example 4: SNP and Mutation Discovery and Validation
The variants from the reference sequence identified by nucleic acid patch PCR and 454 FLX sequencing in Example 2 were validated by performing individual PCR reactions for each variant locus, cloning the amplicons into E. coli, and sequencing 12 clones for each variant. Sequence variants were then analyzed for novelty and whether they affected the translation product of that nucleotide sequence.
Methods
The PCR for each locus in each sample was performed in a total volume of 50 μl. The reaction contained 1×PCR buffer lacking MgCl2 (Invitrogen, Carlsbad, Calif.), 10 units Platinum Taq Polymerase (Invitrogen Carlsbad, Calif.), 0.5 mM each dNTP, 0.5 M Betaine, 0.5 μM Forward Primer, 0.5 μM Reverse Primer, and 100 ng genomic DNA from either the colon tumor or the adjacent normal tissue (Biochain catalog # D8235090-PP-10). This reaction was incubated at 93° C. for 2 minutes, followed by (93° C. for 30 sec, 55° C. for 6 minutes)×30 cycles, and held at 4° C. One fifth of the PCR reaction was verified by electrophoresis on a 2% agarose gel. The PCR products were ligated into the pGEM-T Easy Vector using Rapid Ligation Buffer according to the manufacturer's instructions (Promega, Madison, Wis.), transformed into GC10 Competent Cells (Gene Choice) and grown overnight on LB-agar (Luria-Broth) plates containing standard concentrations of carbenicillin, X-gal and IPTG. After overnight growth, at least 12 colonies were picked from the plates and added to 50 μl colony PCR reactions containing 1×PCR Reaction Buffer (Sigma, St. Louis, Mo.), 2 units Jumpstart Taq Polymerase (Sigma), 0.2 mM each dNTP, 0.5 μM M13 Forward Primer (5′ CGCCAGGGTTTTCCCAGTCACGAC 3′)(SEQ ID NO:383), 0.5 μM M13 Reverse Primer (5′ TCACACAGGAAA CAGCTATGAC 3′)(SEQ ID NO:384), and 0.01% Tween. The reaction was incubated at 94° C. for 10 minutes, followed by (94° C. for 1 min 30 sec, 55° C. for 1 min, 72° C. for 1 min)×35 cycles, and held at 4° C. These reactions were then treated with 10 μl Exo-SAP to degrade the remaining primers and nucleotides by adding 0.2 units Exonuclease I (USB, Cleveland, Ohio) and 0.2 units Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, Wis.) in 1×SAP buffer (Promega, Madison, Wis.), incubating at 37° C. for 30 min, then by 80° C. for 30 min. The Sanger sequencing/cycle sequencing reactions were 20 ul and contained 1.5 μl Exo-SAP Treated colony PCR, 1 μl Big Dye Terminator v3.1 RR-100 Mix (Applied Biosystems, Foster City, Calif.), 2 mM MgCl2, and 0.16 μM M13 Forward Primer. They were incubated at 96° C. for 1 min, followed by (96° C. for 10 sec, 50° C. for 5 sec, 60° C. for 4 min)×24 cycles, and held at 4° C. The reactions were ethanol precipitated with sodium acetate and submitted to the Washington University Genome Sequencing Center to load on the ABI 3730 (Applied Biosystems, Foster City, Calif.). Trace files were analyzed using the Phred software (Ewing and Green 1998; Ewing, Hillier, Wendl and Green 1998), and the resulting sequencing reads were aligned to the reference sequence using the BLAT software on the UCSC Genome Browser (Kent 2002; Kent, Sugnet, Furey, Roskin, Pringle, Zahler and Haussler 2002).
Sequence variants for each exon were identified, and the UCSC Genome browser was used to determine the presence of these variants in the NCBI database of SNPs (dbSNP, www.ncbi.nlm.nih.gov/projects/SNP/index.html), and whether they disrupted a codon. To determine if the tumor specific mutation identified in this analysis had been previously reported, the Catalog of Somatic Mutations in Cancer (www.sanger.ac.uk/genetics/CGP/cosmic/) was searched.
Results
Seven variants from the reference sequence were identified (TABLE E). The SNPs and mutations identified by nucleic acid patch PCR and 454 FLX sequencing were validated by performing individual PCR reactions from the original patient samples, cloning the amplicons, and sequencing at least 8 clones per locus using standard Sanger sequencing. Five of these variants were already in the NCBI database of SNPs (dbSNP; http://www.ncbi.nlm.nih.gov/SNP/). The individual sequenced was germline homozygous at three of these SNPs (rs17883323, rs185587, rs3020646) and was germline heterozygous at two other SNPs in the database, rs2229992 and rs351771. The A allele of the SNP rs2229992 was in 54% of reads from the tumor sample and 54% of reads from normal sample. The C allele of the SNP rs351771 was in 48% of reads from the tumor sample and 47% of reads from normal sample. The ability to detect both alleles of these known polymorphisms at near equal frequency indicates that nucleic acid patch PCR provides high allele sensitivity that is reproducible across samples. SNP in an intron of APC that was not yet in dbSNP (rs62626346) was also discovered. The sequenced individual was heterozygous in both the tumor and normal samples at this intronic position. A novel germline SNP was discovered in the sequenced individual in one of the most extensively surveyed genes, APC. This illustrates that medical resequencing of well-characterized candidate genes will yield more insight into genetic variation in individuals.







TABLE E







Mutation and SNPs discovered. Bold mutation is tumor specific.









Fraction of Reads with Variant






















Colon





Exon

Reference

Amino Acid
Adenocarcinoma
Adjacent Normal


Protein
Ref Seq ID
number
Location*
Base
Variant
Change
Tissue
Tissue




















APC
NM_000038
10
rs2229992
T
C
none
143/301
48%
222/468
47%


APC
NM_000038
12
rs351771
G
A
none
37/68
54%
43/79
54%


APC
NM—000038
12
chr5: 112192485
C
T
Arg−>
23/68
33%
 3/80
 4%








STOP


APC
NM_000038
13
rs62626346 †
T
C
intronic
17/29
59%
27/50
54%


TP53
NM_000546
1
rs17883323
G
T
intronic
41/41
100% 
50/50
100% 


RB1
NM_000321
11
rs185587
G
T
intronic
79/79
100% 
102/102
100% 


RB1
NM_000321
24
rs3020646
C
T
intronic
24/24
100% 
18/18
100% 





*Location is according to the March 2006 human genome assembly from the UCSC Genome Browser


† Novel germline SNP






A tumor-specific nonsense mutation was also discovered. It is a C to T substitution in the APC gene at chr5:112192485 that results in a codon for arginine changing to a stop codon. This is likely a significant mutation in this individual's colon tumor because it is a nonsense mutation in a gene that is already known to cause colon cancer. This mutation was in 33% of reads from the tumor sample. This mutation is adjacent to a heterozygous SNP, and we discovered that 62% of the SNP A allele reads had the nonsense mutation, and 0% of the SNP G allele reads had the nonsense mutation. This indicates that the nonsense mutation occurred on the A allele during the clonal expansion of the tumor. This mutation was previously observed in an ovarian endometrioid adenocarcinoma and is Mutation ID #19040 in the Catalog of Somatic Mutations in Cancer (http://www.sanger.ac.uk/genetics/CGP/cosmic/).
In summary, this method has the allele sensitivity necessary for variant discovery in personal genome sequencing since both alleles of heterozygous SNPs were identified at near-even frequencies. Indeed, the utility of nucleic acid patch PCR is best illustrated by the fact that a novel, cancer-specific mutation was discovered in this pilot study.
Example 5: SNP Sensitivity Analysis
To determine the sensitivity of the nucleic acid patch PCR method coupled with 454 sequencing, each exon analyzed in examples 2 to 5 was individually amplified by PCR from the same colon cancer and adjacent normal tissue samples as used above. Direct Sanger sequencing was then performed. The sequences obtained were then compared to sequences generated using nucleic acid patch PCR and 454 sequencing.
The PCR for each locus in each sample was performed in a total volume of 50 ul. The reaction contained 1×PCR Buffer—MgCl2 (Invitrogen, Carlsbad, Calif.), 5 units Platinum Taq Polymerase (Invitrogen Carlsbad, Calif.), 0.5 mM each dNTP, 0.5 M Betaine, 0.5 μM Locus Specific Forward Primer, 0.5 μM Locus Specific Reverse Primer, and 20 ng genomic DNA from the adjacent normal tissue (Biochain catalog # D8235090-PP-10). This reaction was incubated at 93° C. for 2 min, followed by (93° C. for 30 sec, 55° C. for 6 min)×30 cycles, and held at 4° C. One fifth of the PCR reaction was verified by electrophoresis on a 2% agarose gel. These reactions were then treated with 10 μl Exo-SAP to degrade the remaining primers and nucleotides by adding 0.2 units Exonuclease I (USB, Cleveland, Ohio) and 0.2 units Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, Wis.) in 1×SAP buffer (Promega, Madison, Wis.), incubating at 37° C. for 30 min, then by 80° C. for 30 min. The Sanger sequencing/cycle sequencing reactions were 20 μl and contained 1.5 μl ExoSAP-treated individual exon PCR, 1 μl Big Dye Terminator v3.1 RR-100 Mix (Applied Biosystems, Foster City, Calif.), 2 mM MgCl2, and 0.16 μM Forward or Reverse PCR Primer. They were incubated at 96° C. for 1 min, followed by (96° C. for 10 sec, 50° C. for 5 sec, 60° C. for 4 min)×24 cycles, and held at 4° C. The reactions were ethanol precipitated with sodium acetate and submitted to the Washington University Genome Sequencing Center to load on the ABI 3730 (Applied Biosystems, Foster City, Calif.). Trace files from both forward and reverse reads were analyzed for SNPs using PolyPhred and manual inspection (Nickerson, Tobe and Taylor 1997).
No additional SNPs were identified in the DNA sample beyond the six germline SNPs already identified. Thus, in this experiment, the sensitivity of the method is 100%.
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Example 6: Bisulfite Nucleic Acid Patch PCR Proof of Concept
In this example, various features of the method of the invention are demonstrated including: 1. Creating nucleic acid template with defined ends using AluI restriction digest. 2. Treatment with sodium bisulfite to detect DNA methylation by sequencing. 3. Using small quantities of DNA. The method is depicted in FIG. 4.
Template Preparation
Genomic DNA from breast and colon cancer and adjacent normal tissue was digested with the AluI restriction endonuclease in 10 ul total volume reaction containing genomic DNA, 10 U AluI enzyme (NEB), and 1× NEBuffer 2 (NEB). This reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step. To demonstrate the efficacy of this method with small quantities of DNA, multiple reactions were performed using decreasing quantities of genomic DNA including 900, 675, 450, 250, 225, 112, 70, 50, 20, 1.6, 0.8, and 0.4 ng genomic DNA. A control reaction lacking genomic DNA was also prepared.
Nucleic Acid Patch Ligation
Nucleic acid patch oligos were designed as described in Example 2 but were designed to anneal adjacent to the AluI restriction enzyme site upstream and downstream of promoters of a select 94 gene in the human genome. These loci were selected because they are the promoters of genes frequently mutated in cancer. Nucleic acid patch driven ligation of the universal primers to selected fragments was performed by addition of more reactants to the initial tube to result in the following final concentrations: 2 nM each nucleic acid patch oligo, 200 nM Universal Primer 1, 200 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.
Incorrect products, template genomic DNA and excess primer were degraded as described in Example 2. In brief, 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) were added to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C.
Sodium Bisulfite Treatment
The reactions were then treated with sodium bisulfite to convert unmethylated cytosines to uracil. This was achieved by using the EZ DNA Methylation Gold Bisulfite Treatment Kit (Zymo Research) following the manufacture's instructions. Since the sample volume after the exonuclease treatment was 27 ul, the CT Conversion Reagent from the kit was made by adding 830 ul dH2O instead of 900 ul dH2O. The DNA was eluted from the column in the final step with 10 ul M-Elution buffer.
PCR Amplification
The universal primers were then used to PCR amplify the selected bisulfite converted loci from each sample. For the PCR, reagents were added to the last 10 ul column elution to result in these final concentrations in 50 ul: 0.5 uM each tailed Universal Primer, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl2, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 29 cycles, and held at 4° C. As described in Example 2, the universal PCR used primers tailed with 454 Life Sciences A or B oligo at the 5′ end, followed by a sample specific DNA sequence and ending at the 3′ end with the nucleic acid patch universal primer sequence. The PCR product smear between the expected sizes was confirmed by running on a 3% Metaphor Agarose gel (Lonza). The reactions were then purified on a Qiaquick Spin Column (Qiagen). An aliquot of the reactions was analyzed by gel elctrophoresis on an agrose gel (Lonza).
The eluted DNA of the reactions using 250 ng of genomic DNA was quantified on the Nanodrop (www.nanodrop.com) and the same quantity of DNA was pooled together from each of the separate samples. This pooled sample was submitted for sequencing on the 454 Life Sciences/Roche FLX machine. Sequence and data analysis were as described in Example 2.
Results
Highly multiplexed bisulfite PCR was successful even when small quantities of genomic DNA were used (FIG. 5). The expected smear of products is seen in the lane that contained 900 ng DNA, and the reaction generates the expected products even when as little as 20 ng of genomic DNA is used. Using less than 20 ng of genomic DNA might also have been successful, but the sensitivity of the imaging was not sufficient to reliably detect it.
Sequence analysis of the reactions performed using 250 ng of human tumor genomic DNA demonstrated that 100% of the targeted regions were successfully amplified and sequenced. All of the 94 targeted promoters were sequenced at least once (FIG. 6). The method was also very specific, with 90% of all reads matching the targeted promoters.
In summary, digesting genomic DNA with AluI successfully defined the ends of nucleic acid templates even when a very small quantity of genomic DNA treated with sodium bisulfite was used.
Example 7: Bisulfite Nucleic Acid Patch PCR and Tumor Analysis
Inappropriate CpG DNA methylation has been found in most types of cancers1. Genes that participate in numerous pathways involved in malignancy can acquire aberrant promoter methylation2. Tumor suppressor genes frequently exhibit promoter hypermethylation, an epimutation that is associated with inappropriate gene silencing2. A recent study has found that several key tumor suppressor genes exhibit promoter hypermethylation more often than genetic disruption, suggesting this mechanism is an important driver of tumorigenesis3. Oncogenes can exhibit hypomethylation of their promoters which is associated with inappropriate expression4. More complicated mis-regulation of a gene can also be caused by aberrant methylation; a recent report found that hypermethylation of a p53 binding site blocked binding of the repressor, resulting in overexpression of the survivin oncogenes.
The identification of gene promoters that are aberrantly methylated during tumor development is valuable because it can provide insights into pathways that are commonly disrupted during tumorigenesis that can serve as drug targets6, 7. Analysis of promoter methylation can also classify distinct subtypes of cancers that may have differential clinical characteristics in order to personalize treatment8, 9. Finally, loci that are hypermethylated in tumors are often detected in peripheral samples (e.g. blood or stool) and may serve as diagnostic or prognostic biomarkers10.
Many techniques have been developed to detect DNA methylation including methods based on microarrays11, quantitative PCR12, mass-spectrometry13 and DNA sequencing14. The method that is the most direct and has the highest resolution involves treatment of genomic DNA with sodium bisulfite (which converts unmethylated cytosines to uracil, while leaving methylated cytosines intact) followed by sequencing of single molecules. Not only does this method determine the methylation state at each CpG position across a single molecule, but it also detects sequence variants. This cis information makes it possible to distinguish allele specific methylation14, and is also valuable for quantifying densely methylated molecules in a background of unmethylated or sparsely methylated molecules.
The recent introduction of second-generation DNA sequencing technologies has significantly reduced the cost required to sequence DNA. This has led to several new approaches for studying aberrant methylation using bisulfite PCR and sequencing. Methods for genome-wide surveys of methylation in a small number of samples have been developed including whole genome bisulfite sequencing15, bisulfite sequencing large fractions of restriction digested genomic DNA16, padlock probe based strategies17, 18 and array-based hybridization capture19. In contrast, methods for the detailed study of a few loci across many samples have been described that involve amplifying each locus individually, labeling with sample-specific barcodes and performing ultra-deep bisulfite sequencing20-22. These methods are limited to a small number of loci because the amplification of each locus separately is laborious and requires a significant amount of patient DNA per locus queried. There is still a need for a method that enables the intermediate experiment to be performed. That is, the targeted multiplexed bisulfite PCR and sequencing of an intermediate number of loci (100-1000) across a large number of samples. In cancer research this experiment is crucial since the discoveries made in genome wide profiling of a few samples need to be validated and followed-up across large numbers of patient samples.
We sought to develop a method to perform highly multiplexed bisulfite sequencing across many patient samples simultaneously. Bisulfite treatment significantly reduces the complexity of DNA sequence by converting most Cs to Ts. It also results in molecules from the same locus having different sequences depending on their methylation state. Therefore we perform the oligo hybridization and ligation based selection of the targeted loci before bisulfite treatment. The selection is highly sensitive and specific and only one pair of oligos per locus is needed, even when selecting CpG rich loci. The PCR amplification of selected loci is performed after bisulfite. Therefore the universal primers used to amplify all loci simultaneously had to be designed to exclude C's, so that they would remain unchanged through bisulfite conversion. Since the major application of this method is likely to be in clinical specimens, we optimized the method so that it didn't require large quantities of starting genomic DNA and was compatible with the DNA degradation inherit in sodium bisulfite treatment.
We designed the method to be easy to implement in any lab with standard molecular biology techniques and reagents. We also tested that it would scale up well to process many patient samples in 96-well format. We integrated sample-specific DNA barcodes into the multiplexed amplification so that many patient samples can be pooled and sequenced simultaneously on second-generation sequencing machines. Here we present a proof-of-principle experiment in which we amplified promoter regions from 94 targeted loci simultaneously and sequenced these loci across 48 samples including colon and breast tumor and adjacent normal tissue samples. In this experiment, we characterized the promoter methylation of genes that are known to be frequently mutated in cancer. We identified several novel loci that undergo frequent tumor-specific promoter methylation, and we observed allele-specific methylation patterns that occur during tumor development. We demonstrated that this method utilizes the power of next-generation sequencing to study DNA methylation at many loci across many patient samples.
Results
Overview of Bisulfite Patch PCR
Bisulfite Patch PCR begins with a restriction digest of human genomic DNA to define the ends of the fragments that will be selected (FIGS. 7A & B). Targeted loci are then selected from the genomic restriction fragments by annealing patch oligos to the ends of the targeted genomic fragments. These oligos serve as a patch between the correct fragments and universal primers (U1 & U2) (FIG. 7C). The universal primers are then ligated to the genomic fragments using a thermostable ligase (FIG. 7D). Unselected genomic DNA is then degraded with exonucleases to gain additional selectively (FIG. 7E). Selected fragments are protected from degradation by a 3′ modification on the universal primer U2 (FIG. 7E). Next, the selected fragments are treated with sodium bisulfite to convert unmethylated cytosines to uracil, leaving methylated bases intact (FIG. 7F). The universal primers do not contain cytosine bases so that the sequence remains unchanged through the bisulfite conversion. The bisulfite treated selected fragments are then all amplified together simultaneously by PCR with the universal primers (U1 & U2′) (FIG. 7G). Sample-specific DNA barcodes are incorporated into the universal primers by tailing the 5′ end with a DNA sequence that is specific to each sample and the sequencing platform primers (454 sequencing primers) (FIG. 7G). The final PCR amplicons from each of the samples can be pooled together for sequencing because the first few bases of each sequencing read will identify the sample from which that sequence originated.
Highly Multiplexed Bisulfite Sequencing of CAN Gene Promoters in Colon and Breast Cancer
To test the performance of Bisulfite Patch PCR we analyzed the promoter methlyation of 94 genes that are frequently mutated in breast and colon cancers (′CAN genes′)24. We designed the patch oligos to select AluI restriction digest fragments containing at least three CpG positions within 700 bp upstream of the transcription start site. We chose 42 colon CAN gene promoters, 44 breast CAN gene promoters, 4 gene promoters that were identified as both colon and breast CAN genes, and 4 controls. The four controls include an imprinted locus, a housekeeping gene promoter, and 2 neutral loci that accumulate methylation with mitotic cell division25. These targeted promoter regions ranged in length from 125 bp to 581 bp and totaled 25.4 Kbp (SEQ ID NOs xx-xx). To determine the amount of genomic DNA required for the Bisulfite Patch PCR, we performed gel electrophoresis of the PCR products generated with different amounts of starting DNA. We observed DNA within the expected size range from reactions that started with as much as 1 microgram and as little as 20 nanograms (ng) of human genomic DNA (FIG. 8).
We performed Bisulfite Patch PCR on 250 ng of genomic DNA from each of 48 samples in parallel in a 96-well plate. The genomic DNA was isolated from a panel of 12 colon tumors, 12 matched adjacent normal colon tissues, 12 breast tumors and 12 matched adjacent normal breast tissues (TABLE F). We incorporated a 5-bp sample-specific DNA barcode in the final PCR, pooled the amplicons from all of the samples, and sequenced the pool using the Roche/454 FLX sequencer. We obtained 97,115 reads and aligned these to the in silico bisulfite treated reference sequences of our targeted loci. We successfully amplified all 94 (100%) of the targeted loci, indicating that the method is highly sensitive. Ninety percent (87,458 reads) of all reads mapped to one of the targeted promoters, demonstrating that the method is highly specific. These results demonstrate the Bisulfite Patch PCR enables highly multiplexed bisulfite sequencing.














TABLE F





Lot

Tumor or



DNA
Patient


Number
Tissue
Normal
Age
Sex
Pathological Diagnosis
Barcode
Number






















A811018
Breast
T
34
F
invasive ductal carcinoma
GAGAC
1



Breast
N



GACAT
1


A704203
Breast
T
36
F
invasive ductal carcinoma
GTCGT
2



Breast
N



CAGAT
2


A810202
Breast
T
41
F
invasive ductal carcinoma
AGAGC
3



Breast
N



AGCAT
3


A811022
Breast
T
46
F
invasive ductal carcinoma
GTGTA
4



Breast
N



GTCAC
4


A810219
Breast
T
47
F
invasive ductal carcinoma
ATAGA
5



Breast
N



ATATC
5


A811019
Breast
T
47
F
invasive ductal carcinoma
GACGA
6



Breast
N



GCAGA
6


A810210
Breast
T
48
F
invasive ductal carcinoma
ACGAT
7



Breast
N



ACTAG
7


A810220
Breast
T
48
F
invasive ductal carcinoma
ATCAG
8



Breast
N



ATCGC
8


A811021
Breast
T
50
F
invasive ductal carcinoma
GCTGT
9



Breast
N



GTGAG
9


A810208
Breast
T
55
F
invasive ductal carcinoma
AGCGA
10



Breast
N



ACAGT
10


A810213
Breast
T
58
F
invasive ductal carcinoma,
ACTGC
11







Poorly Differentiated



Breast
N



ACTCT
11


A811020
Breast
T
77
F
invasive ductal carcinoma
GCACG
12



Breast
N



GCTAC
12


B108099
Colon
T
37
M
Adenocarcinoma, mucinous
CTCAT
1



Colon
N



CTCGA
1


A811012
Colon
T
40
M
Adenocarcinoma, Ulcer
TATAC
2



Colon
N



TATGT
2


B105050
Colon
T
52
F
Adenocarcinoma, Moderately
CGTGT
3







Differentiated



Colon
N



CTAGC
3


B105051
Colon
T
56
F
Adenocarcinoma, Ulcer,
CTACT
4







Moderately Differentiated



Colon
N



CTGAC
4


A709116
Colon
T
57
M
Adenocarcinoma, Moderately
CGAGA
5







Differentiated



Colon
N



CGCAG
5


A709121
Colon
T
57
M
Adenocarcinoma, Moderately
CGCGC
6







Differentiated



Colon
N



CGTAC
6


A811013
Colon
T
62
F
Adenocarcinoma
TGCAC
7



Colon
N



TGCGT
7


A811015
Colon
T
65
M
Adenocarcinoma
TGTCG
8



Colon
N



TCAGC
8


A811010
Colon
T
71
F
Adenocarcinoma, Ulcer
TACAG
9



Colon
N



TACGC
9


A811016
Colon
T
75
M
Adenocarcinoma
TCGAC
10



Colon
N



TCGTG
10


A811014
Colon
T
79
M
Adenocarcinoma, Ulcer
TGTAT
11



Colon
N



TGTGA
11


A704198
Colon
T
81
M
Adenocarcinoma, Moderately
CATAG
12







Differentiated



Colon
N



CATGC
12









Coverage of Promoters and Reproducibility

To analyze the uniformity of the sequence coverage, we graphed the number of reads obtained for each targeted promoter versus the length of the targeted region. (FIG. 9A; TABLE G). The abundance of each promoter ranged from 10 to 5114 reads. We calculated that 93% of promoters have coverage within 10 fold of the median coverage (444 reads). There is a strong inverse correlation between amplicon length and the number of reads (linear regression R2=0.42). This correlation indicates that longer amplicons are less abundant in the reaction. If we had restricted our design to a maximum target length of 300 bp, then 92% (57/62) of those promoters would have coverage within 5 fold of the median coverage (1051 reads). These calculations indicate that approximately half of the difference in abundance of the loci is attributable to length bias. While length bias can occur in multiplex PCR, in previous versions of this universal PCR used in nested patch PCR we did not observe a correlation between amplification efficiency and length23. Since the main difference between these methods is the sodium bisulfite treatment, we suspect that longer loci were more likely to be damaged during bisulfite conversion26, and thus are less abundant in the reaction.
To test if bisulfite patch PCR reproducibly amplifies selected loci, we calculated the number of reads per locus in each of the 48 samples that were prepared in parallel. We then calculated the correlation coefficient, r, for the number of reads per locus between all possible pairs of samples. The histogram of r values obtained for the pair-wise correlations between all 48 samples is depicted in FIG. 9B. The mean r value is 0.91, indicating that the number of reads per locus is highly reproducible across patient samples. This indicates that the abundance of each locus in the reaction is not stochastic, but represents something intrinsic to the locus, including the length, as discussed above.

















TABLE G









Length
Number











of
of CGs


CAN



Ampli-
per


Gene


# of
con
Ampli-
Meth-


Type
Gene
Accession
Reads
(bp)
con
ylated
BT
BN
CT
CN

























Breast
DPYD
NM_000110
1207
214
6
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
XDH
NM_000379
313
276
3
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
CYP1A1
NM_000499
478
259
23
N
(0%)0/12
(0%)0/12
(0%)0/11
(0%)0/12


Breast
DPAGT1
NM_001382
278
182
7
N
(0%)0/12
(0%)0/11
(0%)0/12
(0%)0/10


Breast
CLCN3
NM_001829
2405
163
15
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
MYH9
NM_002473
167
368
31
N
(0%)0/11
(0%)0/12
(0%)0/12
(0%)0/12


Breast
PRPF4B
NM_003913
997
225
10
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
TIMELESS
NM_003920
12
308
17
N
(0%)0/4 
(0%)0/2 
(0%)0/2 
(0%)0/2 


Breast
LRRFIP1
NM_004735
464
165
14
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
NUP214
NM_005085
1963
201
12
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
TLN1
NM_006289
282
297
22
N
(0%)0/12
(0%)0/11
(0%)0/12
(0%)0/12


Breast
ABCB8
NM_007188
2390
179
8
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
ZNF646
NM_014699
2451
202
7
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
PDCD11
NM_014976
891
246
17
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
MAPKBP1
NM_014994
221
382
26
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
C14orf100
NM_016475
556
287
34
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
NOTCH1
NM_017617
81
282
34
N
(0%)0/9 
(0%)0/10
(0%)0/12
(0%)0/7 


Breast
SULF2
NM_018837
2094
211
21
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
KIAA0999
NM_025164
682
252
13
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
PLEKHA8
NM_032639
71
334
40
N
(0%)0/9 
(0%)0/10
(0%)0/7 
(0%)0/9 


Breast
FLJ40869
NM_182625
385
245
31
N
(0%)0/11
(0%)0/12
(0%)0/12
(0%)0/12


Breast
TMEM123
NM_052932
329
383
11
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
KIAA0427
NM_014772
450
220
15
N
(8%)1/12
(8%)1/12
(17%)2/12 
(8%)1/12


Breast
VEPH1
NM_024621
1563
210
5
N
(8%)1/12
(8%)1/12
(8%)1/12
(0%)0/12


Breast
SLC8A3
NM_182932
70
304
9
N
(0%)0/10
(0%)0/8 
(0%)0/11
(0%)0/10


Breast
RGL1
NM_015149
10
581
60
N
(0%)0/2 
(0%)0/3 
(0%)0/3 
(0%)0/2 


Colon
ERCC6
NM_000124
1784
171
15
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
NF1
NM_000267
293
198
19
N
(0%)0/12
(0%)0/11
(0%)0/12
(0%)0/12


Colon
PTEN
NM_000314
111
412
20
N
(0%)0/11
(0%)0/9 
(0%)0/12
(0%)0/9 


Colon
GALNS
NM_000512
1005
242
26
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
GUCY1A2
NM_000855
37
313
15
N
(0%)0/6 
(17%)1/6 
(0%)0/4 
(0%)0/7 


Colon
UQCRC2
NM_003366
610
163
10
N
(0%)0/12
(0%)0/11
(0%)0/12
(0%)0/12


Colon
MCM3AP
NM_003906
105
488
20
N
(0%)0/8 
(0%)0/11
(0%)0/8 
(0%)0/11


Colon
EPHB6
NM_004445
1842
172
13
N
(0%)0/11
(0%)0/11
(0%)0/12
(0%)0/12


Colon
KRAS
NM_004985
18
415
53
N
(0%)0/3 
(0%)0/5 
(0%)0/4 
(0%)0/3 


Colon
ZNF262
NM_005095
359
302
21
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
SMAD4
NM_005359
634
217
21
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
SFRS6
NM_006275
402
338
35
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
SMTN
NM_006932
145
397
34
N
(0%)0/12
(0%)0/12
(0%)0/11
(0%)0/10


Colon
KIAA0556
NM_015202
281
353
38
N
(0%)0/11
(0%)0/11
(0%)0/12
(0%)0/12


Colon
ADARB2
NM_018702
26
374
7
N
(0%)0/2 
(17%)1/6 
(0%)0/6 
(0%)0/5 


Colon
FBXW7
NM_033632
1097
204
24
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
DTNB
NM_183361
2195
189
19
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
RET
NM_020975
58
412
46
N
(0%)0/6 
(0%)0/8 
(0%)0/6 
(0%)0/8 


Colon
KIAA0367
NM_015225
170
235
18
N
(8%)1/12
(0%)0/11
(8%)1/12
(0%)0/10


Colon
SH3TC1
NM_018986
2876
157
10
N
(8%)1/12
(0%)0/12
(0%)0/12
(0%)0/12


Colon
TIAM1
NM_003253
354
283
40
N
(0%)0/12
(0%)0/12
(8%)1/12
(0%)0/12


Colon
C13orf7
NM_024546
351
269
10
N
(0%)0/11
(8%)1/12
(17%)2/12 
(0%)0/12


Control
HSP
NM_007355
155
381
21
N
(0%)0/11
(0%)0/12
(0%)0/10
(0%)0/11


Dual
TP53
NM_000546
1132
154
6
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Dual
PIK3CA
NM_006218
989
270
15
N
(0%)0/12
(0%)0/12
(0%)0/12
(0%)0/12


Breast
TECTA
NM_005422
168
393
7
Y
(100%)11/11 
(100%)11/11 
(100%)12/12 
(91%)10/11


Breast
KIAA0467
NM_015284
332
267
6
Y
(100%)12/12 
(100%)12/12 
(92%)11/12
(100%)12/12 


Breast
RP1L1
NM_178857
416
221
7
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(100%)12/12 


Breast
LOC340156
NM_001012418
1481
175
3
Y
(100%)12/12 
(100%)12/12 
(83%)10/12
(100%)12/12 


Breast
DBN1
NM_004395
3750
165
3
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(92%)11/12


Breast
CENTG1
NM_014770
3613
190
3
Y
(100%)12/12 
(100%)12/12 
(92%)11/12
(100%)12/12 


Breast
KIAA1946
NM_177454
1667
212
5
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(100%)12/12 


Breast
CMYA1
NM_194293
127
173
4
Y
(100%)11/11 
(83%)8/9 
(83%)10/12
(70%)7/10 


Breast
AEGP
NM_206920
902
221
6
Y
(83%)10/12
(100%)12/12 
(83%)10/12
(100%)12/12 


Breast
TAF1
NM_004606
2315
153
12
Y
(58%)7/12 
(67%)8/12 
(25%)3/12 
(25%)3/12 


Breast
RPGRIP1
NM_020366
48
562
7
Y
(88%)7/8 
(100%)7/7  
(100%)9/9  
(43%)3/7 


Breast
SLC9A10
NM_183061
2096
195
3
Y
(100%)12/12 
(100%)12/12 
(33%)4/12 
(75%)9/12 


Breast
COL19A1
NM_001858
3069
168
4
Y
(58%)7/12 
(58%)7/12 
(33%)4/12 
(8%)1/12


Breast
ABP1
NM_001091
200
207
5
Y
(36%)4/11 
(67%)8/12 
(64%)7/11 
(58%)7/12 


Breast
CSPP1
NM_024790
476
330
3
Y
(17%)2/12 
(42%)5/12 
(15%)3/12 
(67%)8/12 


Breast
NCB5OR
NM_016230
282
434
6
Y
(42%)5/12 
(17%)2/12 
(50%)6/12 
(18%)2/11 


Colon
ITGAE
NM_002208
348
349
9
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(100%)12/12 


Colon
TGM3
NM_003245
178
418
5
Y
(100%)10/10 
(100%)12/12 
(100%)11/11 
(100%)10/10 


Colon
DSCAML1
NM_020693
2052
205
7
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(100%)12/12 


Colon
TNN
NM_022093
2659
161
3
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(100%)12/12 


Colon
ACSL5
NM_016234
1280
235
5
Y
(100%)12/12 
(100%)12/12 
(83%)10/12
(92%)11/12


Colon
SEC8L1
NM_021807
221
345
4
Y
(82%)9/11 
(100%)10/10 
(100%)12/12 
(100%)12/12 


Colon
PCDHA9
NM_014005
1761
247
4
Y
(83%)10/12
(92%)11/12
(83%)10/12
(100%)12/12 


Colon
C1QR1
NM_012072
1646
172
8
Y
(100%)12/12 
(83%)10/12
(67%)8/12 
(50%)6/12 


Colon
STAB1
NM_015136
46
511
16
Y
(86%)6/7 
(100%)9/9  
(70%)7/10 
(50%)4/6 


Colon
HAPLN1
NM_001884
108
371
11
Y
(91%)10/11
(83%)5/6 
(30%)3/10 
(55%)6/11 


Colon
BCL9
NM_004326
266
256
5
Y
(67%)8/12 
(64%)7/11 
(0%)0/12
(25%)3/12 


Colon
SCN3B
NM_018400
98
382
11
Y
(27%)3/11 
(13%)1/8 
(0%)0/11
(10%)1/10 


Colon
GPR158
NM_020752
125
315
37
Y
(8%)1/12
(11%)1/9 
(27%)3/11 
(0%)0/9 


Colon
HIST1H1B
NM_005322
962
208
3
Y
(25%)3/12 
(17%)2/12 
(25%)3/12 
(8%)1/12


Colon
NUP210
NM_024923
68
419
24
Y
(0%)0/8 
(25%)2/8 
(11%)1/9 
(20%)2/10 


Control
NKX2-5
NM_004387
1980
184
10
Y
(100%)12/12 
(100%)12/12 
(100%)12/12 
(92%)11/12


Control
SOX10
NM_006941_1
1170
213
22
Y
(100%)12/12 
(67%)8/12 
(100%)12/12 
(100%)12/12 


Control
H19
AK311497
1614
177
9
Y
(58%)7/12 
(83%)10/12
(75%)9/12 
(92%)11/12


Breast
ICAM5
NM_003259
1717
178
11
Y
(33%)4/12 
(8%)1/12
(58%)7/12 
(0%)0/12


Breast
PPM1E
NM_014906
2932
170
10
Y
(25%)3/12 
(0%)0/12
(42%)5/12 
(0%)0/12


Colon
IGFBP3
NM_000598
791
276
25
Y
(67%)8/12 
(8%)1/12
(75%)9/12 
(25%)3/12 


Colon
UHRF2
NM_152896
800
185
10
Y
(58%)7/12 
(8%)1/12
(50%)6/12 
(8%)1/12


Colon
KCNQ5
NM_019842
181
273
21
Y
(0%)0/10
(0%)0/12
(92%)11/12
(33%)4/12 


Colon
CLSTN2
NM_022131
80
304
35
Y
(0%)0/8 
(10%)1/10 
(56%)5/9 
(0%)0/10


Colon
APC
NM_000038
42
395
16
Y
(29%)2/7 
(0%)0/3 
(0%)0/8 
(0%)0/6 


Dual
LAMA1
NM_005559
438
169
14
Y
(50%)6/12 
(8%)1/12
(67%)8/12 
(17%)2/12 


Dual
SORL1
NM_003105
5114
125
3
Y
(33%)4/12 
(92%)11/12
(0%)0/12
(42%)5/12 









Allele Sensitivity at Imprinted Locus

We next sought to determine if methylated and unmethylated molecules from the same locus are amplified with similar efficiencies. This is requisite if the method is to be used to make quantitative measurements of promoter methylation. The imprinted region from the H19 locus (AK311497), which was included as a control, allows the direct comparison of the amplification efficiency of methylated and unmethylated alleles. We identified nine patients in our panel who were heterozygous for a SNP (rs2251375) in the H19 locus. We used this SNP to identify allele-specific methylation and to quantify the number of sequencing reads obtained for each allele. Allele specific methylation was observed, and both alleles were amplified with nearly equal efficiencies (FIG. 10). Imprinting methylation was observed on either allele in different individuals, consistent with the parent-of-origin determining which allele is methylated, and both alleles were represented at similar frequencies—on average 42% of the sequencing reads corresponded to the ‘G’ allele, 58% to the ‘T’ allele. Thus, our method amplifies methylated and unmethylated molecules from the same locus with nearly equal efficiency, which is crucial for quantifying heterogeneous methylation within tumors.
CAN Gene Promoter Methylation
We next examined the methylation patterns found at the targeted CAN gene promoters to determine if they exhibited tumor specific methylation. Since these genes were previously shown to be frequently mutated in colon and breast tumors24, we hypothesized that the promoters of these genes might also be frequently hyper- or hypomethylated in these cancers. (TABLE H, Detailed in TABLE G).
Approximately half, (51/94), of all the promoters were unmethylated in all tissue types that we tested, including, the negative control promoter of the housekeeping gene HSP90AB1 (NM_007355). Approximately one third, (34/94), of all promoters were methylated in both cancer and normal tissue including all 3 (100%) of the positive control gene promoters, the H19 imprinted promoter (AK311497) and two neutral loci that accumulated DNA methylation with mitotic division (NM_006941 Exon 2, and NM_004387 3′ UTR)25. The remaining nine promoters exhibited tumor-specific aberrant methylation.












TABLE H






Colon
Breast
Dual





CAN
CAN
CAN
Con-




genes
genes
genes
trols
Total




















Unmethylated
22
26
2
1
51


Methylated In Tumor and
15
16
0
3
34


Normal Tissues







Tumor Specific Methylation:
2
2
2
0
6


Breast & Colon







Tumor Specific Methylation:
2
0
0
0
2


Colon







Tumor Specific Methylation:
1
0
0
0
1


Breast







Total
42
44
4
4
94









Tumor Specific Promoter Methylation

Of the nine promoters that exhibited tumor specific methylation, 5 were promoters from colon CAN genes, 2 were promoters from breast CAN genes, and 2 were promoters from genes that were frequently mutated in both colon and breast cancer (‘dual CAN genes’) (TABLE H, Detailed in TABLE G).
Five promoters exhibited tumor-specific hypermethylation in both breast and colon tumors (IGFBP3, UHRF2, LAMA1, ICAM5, PPM1E). One promoter (SORL1) exhibited tumor-specific hypomethylation in both types of cancer. The methylation patterns of ICAM5 and LAMA1 are depicted in FIG. 11 Panels A and B, respectively. Tumor specific promoter methylation of ICAM53 and IGFBP327 was recently reported in different cohorts of breast and colon cancers. The other three loci are novel observations of aberrant tumor methylation. The frequent hypermethylation of these five loci in both types of tumors indicates that common molecular defects are shared between colon and breast cancer. The molecular defect could be an error in both types of tumors that directs methylation to these loci or it could suggest that the inactivation of these genes is a key step in tumorigenesis in both tissues.
These five loci that are hypermethylated in both breast and colon cancer are methylated in 25% to 75% of tumors (TABLE I). Loci that exhibit frequent tumor-specific methylation are often useful as clinical biomarkers. A valuable biomarker would occur frequently in patients' tumors and would be easily distinguished from normal samples. We calculated the sensitivity and specificity of these loci across our samples. The presence of aberrant methylation at two or more of these five methylated markers is found in 9 out of 12 breast tumors (75%), 11 out of 12 colon tumors (92%), 1 of 12 normal breast (8%) and 1 of 12 normal colon (8%). These strong classifiers of cancer vs. normal samples are good candidates for follow-up studies to evaluate their potential as biomarkers for stratifying disease subtypes or as diagnostic biomarkers that can be detected in peripheral specimens. The frequency of aberrant methylation at these loci approaches the significance of even the most common genetic mutations such as APC or TP53 mutations, which are reported to occur in 40-80% of tumors28. This supports the previously proposed hypothesis that epigenetic defects at CAN genes may be more frequent than genetic mutations3.
Three of the CAN gene promoters show tumor specific methylation in only one type of cancer. Colon tumor specific methylation was found in the promoters of KCNQ5 (NM_019842) and CLSTN2 (NM_022131), and those methylation patterns are depicted in FIG. 11 Panels C and D, respectively. Breast tumor specific methylation was found in the promoter of APC (NM_000038). The frequency of these aberrant events in each tumor type is cataloged in TABLE I and suggests that these loci may represent frequent tumor-specific epimutations which merit follow up investigation in a larger cohort of tumors, adjacent normal and cancer-free patient's tissue.







TABLE I







Promoters Exhibiting Tumor Specific Methylation











Gene promoters exhibiting tumor specific hyper- methylation in both breast and colon tumors are not indicated by an * or **.


*Gene promoters exhibiting tumor specific hyper- methylation in one tumor type.


**Gene promoters exhibiting tumor specific hypo- methylation in both breast and colon tumors.






Allelic Tumor Methylation

The single molecule resolution of bisulfite sequencing allows us to simultaneously assess methylation status and identify single nucleotide polymorphisms (SNPs). As seen in FIG. 12, we can distinguish whether tumor specific methylation is occurring on one allele or on both alleles in individuals that are heterozygous for the SNP (rs2854744) in IGFBP3 (NM_000598). Although some aberrant promoter methylation events are known to always occur on both alleles, such as MLH1 promoter methylation29, we found examples in which aberrant methylation was observed on only one allele: Breast Cancer Patient 4 acquired tumor-specific methylation primarily on the A allele, while Colon Cancer Patient 6 acquired tumor-specific methylation primarily on the C allele (FIG. 12). However, other patients acquired aberrant methylation on both alleles during tumorigenesis, such as Breast Cancer Patient 6 and Colon Cancer Patient 7 (FIG. 12). If associated with silencing, this bi-allelic methylation would indicate that both copies of the gene are inactive. Some patients exhibit different allelic methylation patterns between their tumor and adjacent normal tissue: Colon Cancer Patient 12 has methylation on their A allele across all CpGs in both the tumor and the adjacent normal tissue, but as the tumor formed the C allele acquired methylation, specifically in the region of the promoter most distal from the SNP. This suggests that the accumulation of methylation on each allele can occur in different regions of the locus and can occur at different times in tumor development. This type of allelic analysis is useful for resolving intra-tumor heterogeneity of DNA methylation, identifying heterozygous and homozygous epimutations, and understanding the accumulation of aberrant DNA methylation in different tumors.
Discussion
We have developed a method to perform highly multiplexed bisulfite sequencing of many loci across many patient samples simultaneously. This method is highly sensitive and specific and integrates sample specific DNA barcodes into the library construction so that many samples can be pooled to fully utilize the power of next-generation sequencing. Many methods are being developed to perform genome-wide profiling of DNA methylation in individual samples. Bisulfite Patch PCR provides an efficient workflow to utilize second-generation sequencing to follow up and validate aberrant methylation at many loci across large numbers of samples.
In this proof-of-principle experiment, we applied this method to characterize the promoter methylation of genes that are frequently mutated in cancer. From the 94 gene promoters that we analyzed we found that approximately 10% showed tumor specific DNA methylation in breast or colon cancer when compared to adjacent normal tissue. Our data support the previously proposed hypothesis that a relatively small set of genes that are important for tumorigenesis are disrupted in multiple ways in cancers, including frequent epigenetic defects3. We found five loci that can be used to classify tumor and normal samples with high sensitivity (9/12 breast tumors, 11/12 colon tumors) and high specificity (1/12 adjacent normal breast tissues, 1/12 adjacent normal colon tissues). In some samples we observed very low-frequency methylation of these loci in the adjacent normal tissue that may represent a field defect surrounding the tumor, or it may be a part of normal variation between individuals. Follow-up studies that include larger cohorts, cancer-free control patients and peripheral samples from patients with cancer will help determine if these new molecular defects can be useful biomarkers in the clinic. We also utilized SNPs in the sequencing data to observe allele-specific methylation patterns that provide insights into the accumulation of aberrant DNA methylation during tumor development. This method would be valuable for comparing the allelic accumulation of methylation across tumors with different stages and grades to understand the timing of aberrant methylation.
The method presented here fills a gap in the arsenal of tools for the characterization of aberrant DNA methylation. It provides the high resolution of bisulfite sequencing with the throughput of sampling many loci across many samples. This enables an experimental scale that promises to be useful in the effort to understand cancer.
Methods
Design of Patch Oligonucleotides
Human promoter sequence between the transcription start site (TSS) and 700 bp upstream of the TSS was downloaded from the March 2006 assembly on the UCSC Genome Browser (www.genome.ucsc.edu) for the RefSeq genes listed in SEQ ID NOs 582-675. These sequences were then scanned for AluI restriction enzyme recognition sequences, and AluI restriction fragments that were between 125 bp and 600 bp in length and containing at least 3CpG positions were selected. A patch oligo was then designed by sequentially including base pairs from the AluI restriction site into the fragment sequence until the Tm of the patch oligo was between 62° C. and 67° C. Any fragment whose patch oligos contained repetitive elements according to the repeat masker track on the UCSC Genome Browser (www.genome.ucsc.edu) were excluded. The patch oligos were then appended with the complement universal primer sequences to result in the appropriate patch sequence. Patch oligonucleotides were synthesized by SigmaGenosys http://www.sigmaaldrich.com/Brands/Sigma_Genosys.html). Ninety-four pairs of patch oligos were ordered in a 96-well plate. The patch oligos for two loci were duplicated on the plate so that when equimolar portions were pooled from each well these two loci were twice as concentrated in the pool. This was used to measure how the concentration of patch oligos affected amplification efficiency during protocol development. Two universal primer sequences were synthesized by IDT (www.idtdna.com), including U2, which has a 5′ phosphate and a 3 carbon spacer on the 3′ end. Oligonucleotide sequences are listed in TABLE J.







TABLE J







Patch Oligonucleotide Sequences Naming: Refseq 


Accession Number of Locus, L (left) or R (right) side









SEQ ID

Sequence (universal sequence and Alul restricition


NO:
Oligo Name
site in capitals)





385
NM_000110 L
taggtgggcggggtttgAGATCACCAACTACCCACACACACC


 


386
NM_015149 L
gcaccggcgcggAGATCACCAACTACCCACACACACC


 


387
NM_015284 L
ttgcccacctggagagcAGATCACCAACTACCCACACACACC


 


388
NM_182625 L
ggggagaggtctggggaaAGATCACCAACTACCCACACACACC


 


389
NM_000379 L
attctcagagtcactgctaatagAGATCACCAACTACCCACACACACC


 


390
NM_177454 L
gcatcaccgccatcattgcttAGATCACCAACTACCCACACACACC


 


391
NM_004735 L
cctcaggccacgctgAGATCACCAACTACCCACACACACC


 


392
NM_194293 L
ggggaaacagagggggagaAGATCACCAACTACCCACACACACC


 


393
NM_183061 L
gggacagtggatttctgacaaagAGATCACCAACTACCCACACACACC


 


394
NM_024621 L
ctttttttcgttatttgctgggaAGATCACCAACTACCCACACACACC


 


395
NM_001829 L
cagcgtccgggagcAGATCACCAACTACCCACACACACC


 


396
NM_004395 L
ccattctcagcccctacccAGATCACCAACTACCCACACACACC


 


397
NM_001012418 L
tgtcaatactctcggatttacaaAGATCACCAACTACCCACACACACC


 


398
NM_003913 L
aatgcttaaccatctcgctagacAGATCACCAACTACCCACACACACC


 


399
NM_001858 L
ggtaattggctttttaacggttgAGATCACCAACTACCCACACACACC


 


400
NM_016230 L
cactgggaattgtgtactgatgcAGATCACCAACTACCCACACACACC


 


401
NM_032639 L
tctagtccctattcttgttccaaAGATCACCAACTACCCACACACACC


 


402
NM_001091 L
gaaggacttggctgggagaaAGATCACCAACTACCCACACACACC


 


403
NM_007188 L
ccgactggccctccaAGATCACCAACTACCCACACACACC


 


404
NM_024790 L
gaaagtcagtgccaaaacagcaAGATCACCAACTACCCACACACACC


 


405
NM_178857 L
ggaggcccgaaagaagcAGATCACCAACTACCCACACACACC


 


406
NM_005085 L
ttagatgtaggttggctattggtAGATCACCAACTACCCACACACACC


 


407
NM_017617 L
cgggcggggagcAGATCACCAACTACCCACACACACC


 


408
NM_006289 L
gtgcccgaggcctacAGATCACCAACTACCCACACACACC


 


409
NM_206920 L
aggactcaaccagtccagcAGATCACCAACTACCCACACACACC


 


410
NM_004606 L
cgtaaattatacaggcattcccgAGATCACCAACTACCCACACACACC


 


411
NM_014976 L
cctcttttcttctgtatgtccatAGATCACCAACTACCCACACACACC


 


412
NM_052932 L
tgctcagaactctgaagtgacatAGATCACCAACTACCCACACACACC


 


413
NM_025164 L
cttgaggccacaaatgcaggaatAGATCACCAACTACCCACACACACC


 


414
NM_001382 L
cacaactcagttcccggaaacaaAGATCACCAACTACCCACACACACC


 


415
NM_005422 L
ctggatttcctaattttcactacAGATCACCAACTACCCACACACACC


 


416
NM_003920 L
gttttatttgggaggaagtaaagAGATCACCAACTACCCACACACACC


 


417
NM_014770 L
tacgatgtaaccctttttcaggcAGATCACCAACTACCCACACACACC


 


418
NM_020366 L
tagaactactatgtaaacttgggAGATCACCAACTACCCACACACACC


 


419
NM_182932 L
ttgtgagagacgcttgggtgAGATCACCAACTACCCACACACACC


 


420
NM_016475 L
ggtcctagtcccgagcgAGATCACCAACTACCCACACACACC


 


421
NM_014994 L
ggcccgagggaccgtAGATCACCAACTACCCACACACACC


 


422
NM_00499  L
cagagcccgggcgactAGATCACCAACTACCCACACACACC


 


423
NM_014699 L
cgggaactttcccttccttcctAGATCACCAACTACCCACACACACC


 


424
NM_014906 L
ctaccctcacgtggttaagagtgAGATCACCAACTACCCACACACACC


 


425
NM_014772 L
tgtgctaatggcagatgaaaaggAGATCACCAACTACCCACACACACC


 


426
NM_003259 L
ctggctgagatgccatgataataAGATCACCAACTACCCACACACACC


 


427
NM_018837 L
gccgcgacccgcAGATCACCAACTACCCACACACACC


 


428
NM_002473 L
tcggggcgcggagAGATCACCAACTACCCACACACACC


 


429
NM_005095 L
caagtctctttgctgccagcAGATCACCAACTACCCACACACACC


 


430
NM_004326 L
aaaggaaaaagcaaagtcccattAGATCACCAACTACCCACACACACC


 


431
NM_022093 L
ccacacgccaacagtacaagAGATCACCAACTACCCACACACACC


 


432
NM_183361 L
ccccgtgaactccgcaAGATCACCAACTACCCACACACACC


 


433
NM_024923 L
ctcagccagagagccccaAGATCACCAACTACCCACACACACC


 


434
NM_015136 L
cagcccatgctcagccAGATCACCAACTACCCACACACACC


 


435
NM_022131 L
ctccactccgactctcggaaaAGATCACCAACTACCCACACACACC


 


436
NM_006218 L
ttctacgagcagcaggcgAGATCACCAACTACCCACACACACC


 


437
NM_018986 L
ccgcagccggttgatcattAGATCACCAACTACCCACACACACC


 


438
NM_033632 L
cacgggacgaggcagaAGATCACCAACTACCCACACACACC


 


439
NM_001884 L
acaatgatgatagtggcacataaAGATCACCAACTACCCACACACACC


 


440
NM_000038 L
gaattaaaaatagttaccagaaaAGATCACCAACTACCCACACACACC


 


441
NM_014005 L
cttctgtccttgattactgcaggAGATCACCAACTACCCACACACACC


 


442
NM_005322 L
caagtaacacaggcacaggacAGATCACCAACTACCCACACACACC


 


443
NM_019842 L
ctggcaggggctttgcAGATCACCAACTACCCACACACACC


 


444
NM_021807 L
attgatgaagaaaagacagtataAGATCACCAACTACCCACACACACC


 


445
NM_000598 L
cattcgtgtgtacctcgtggAGATCACCAACTACCCACACACACC


 


446
NM_004445 L
ctaaaacagtggggctcctactcAGATCACCAACTACCCACACACACC


 


447
NM_015225 L
ccgggggaggcactcAGATCACCAACTACCCACACACACC


 


448
NM_152896 L
caccgcgctcaacaggaaAGATCACCAACTACCCACACACACC


 


449
NM_018702 L
acaatgacacaaaaggaagagaaAGATCACCAACTACCCACACACACC


 


450
NM_020752 L
gaggaaagccagtttaaagaggcAGATCACCAACTACCCACACACACC


 


451
NM_000314 L
ggctcgtttgccctaaaaatgaaAGATCACCAACTACCCACACACACC


 


452
NM_016234 L
caggggggccctggAGATCACCAACTACCCACACACACC


 


453
NM_020975 L
caggaggcggggaagAGATCACCAACTACCCACACACACC


 


454
NM_000124 L
gcgagcagggcgagaaAGATCACCAACTACCCACACACACC


 


455
NM_000855 L
cccatcctgctggagcAGATCACCAACTACCCACACACACC


 


456
NM_020693 L
tgtcttcacctacccacccctatAGATCACCAACTACCCACACACACC


 


457
NM_018400 L
attagccactccctagtcctagcAGATCACCAACTACCCACACACACC


 


458
NM_024546 L
cacgtttcaatttttttcaaaacAGATCACCAACTACCCACACACACC


 


459
NM_003366 L
ggctacatagaatataaaaacttAGATCACCAACTACCCACACACACC


 


460
NM_015202 L
cgcacccgggcatcAGATCACCAACTACCCACACACACC


 


461
NM_000512 L
aggaggccttcgccgAGATCACCAACTACCCACACACACC


 


462
NM_002208 L
cacagaacacgccgttgacAGATCACCAACTACCCACACACACC


 


463
NM_000267 L
ctggcgctgggctcAGATCACCAACTACCCACACACACC


 


464
NM_005559 L
gattccgagaaactatgtgcccAGATCACCAACTACCCACACACACC


 


465
NM_005359 L
caaggagcgcgggagAGATCACCAACTACCCACACACACC


 


466
NM_003245 L
ccacccctctcaactcacaaAGATCACCAACTACCCACACACACC


 


467
NM_012072 L
ggggctaggaactcgaggaAGATCACCAACTACCCACACACACC


 


468
NM_006275 L
tctttcttggagccctggcAGATCACCAACTACCCACACACACC


 


469
NM_003253 L
agggagcccctaacaaagcAGATCACCAACTACCCACACACACC


 


470
NM_003906 L
gggcgctgccacgaAGATCACCAACTACCCACACACACC


 


471
NM_006932 L
ccctttctcgcgtcagtgtttaAGATCACCAACTACCCACACACACC


 


472
NM_004985 L
CTGACCGGTCTCCACAGAGAAGATCACCAACTACCCACACACACC


 


473
NM_007355 L
ccgaaaaagagcggaggcAGATCACCAACTACCCACACACACC


 


474
AK311497 L
gattcccatccagttgaccgAGATCACCAACTACCCACACACACC


 


475
NM_004387 L
CCCCCGAGAGTCAGGGAGATCACCAACTACCCACACACACC


 


476
NM_006941_1 L
CTCCTTCTTGACCTTGCCCAGATCACCAACTACCCACACACACC


 


477
NM_005559 L
gattccgagaaactatgtgcccAGATCACCAACTACCCACACACACC


 


478
NM_006218 L
ttctacgagcagcaggcgAGATCACCAACTACCCACACACACC


 


479
NM_003105 L
ACAGCAAAAACTACCCTTGATCAAGATCACCAACTACCCACACACACC


 


480
NM_000546 L
GGTGGAAAATTCTGCAAGCCAGAGATCACCAACTACCCACACACACC


 


481
NM_000110 R
CTACCCCACCTTCCTCATTCTCTCTaggcaggcggggc


 


482
NM_015149 R
CTACCCCACCTTCCTCATTCTCTCTtttggccctccctctcg


 


483
NM_015284 R
CTACCCCACCTTCCTCATTCTCTCTtaccttgtgccgggcc


 


484
NM_182625 R
CTACCCCACCTTCCTCATTCTCTCTgcggcggtgttcatgg


 


485
NM_000379 R
CTACCCCACCTTCCTCATTCTCTCTtcagggcatgaagagttcttgg


 


486
NM_177454 R
CTACCCCACCTTCCTCATTCTCTCTggtagaccctcacagcgtc


 


487
NM_004735 R
CTACCCCACCTTCCTCATTCTCTCTccacccgcagggg


 


488
NM_194293 R
CTACCCCACCTTCCTCATTCTCTCTgcctttatcttgctggctagtg


 


489
NM_183061 R
CTACCCCACCTTCCTCATTCTCTCTtcaggcccatcatctcttactt


 


490
NM_024621 R
CTACCCCACCTTCCTCATTCTCTCTtcattaacacttccctctccct


 


491
NM_001829 R
CTACCCCACCTTCCTCATTCTCTCTcacgtcagtcactcacgca


 


492
NM_004395 R
CTACCCCACCTTCCTCATTCTCTCTtcagccccatgcttagcac


 


493
NM_001012418 R
CTACCCCACCTTCCTCATTCTCTCTgttgccttcttagtcagatggg


 


494
NM_003913 R
CTACCCCACCTTCCTCATTCTCTCTcttcagtcaatgctagaaatgg


 


495
NM_001858 R
CTACCCCACCTTCCTCATTCTCTCTgggagtaatgcctttcaggttt


 


496
NM_016230 R
CTACCCCACCTTCCTCATTCTCTCTgttccttagccttggtgctga


 


497
NM_032639 R
CTACCCCACCTTCCTCATTCTCTCTgccggtcgcaggc


 


498
NM_001091 R
CTACCCCACCTTCCTCATTCTCTCTgacagatggaccagggcag


 


499
NM_007188 R
CTACCCCACCTTCCTCATTCTCTCTgtgattggaggatatgttgtca


 


500
NM_024790 R
CTACCCCACCTTCCTCATTCTCTCTtaggaacagtgtaagagcctgg


 


501
NM_178857 R
CTACCCCACCTTCCTCATTCTCTCTcccaccctgttccagttgt


 


502
NM_005085 R
CTACCCCACCTTCCTCATTCTCTCTcgggctgagtagtggc


 


503
NM_017617 R
CTACCCCACCTTCCTCATTCTCTCTgagccgcgcgtcc


 


504
NM_006289 R
CTACCCCACCTTCCTCATTCTCTCTtggggtagaaggcggag


 


505
NM_206920 R
CTACCCCACCTTCCTCATTCTCTCTcccacctgcccgg


 


506
NM_004606 R
CTACCCCACCTTCCTCATTCTCTCTgctcgagtcacgtggctta


 


507
NM_014976 R
CTACCCCACCTTCCTCATTCTCTCTagaaaaaacgaggggcgcaag


 


508
NM_052932 R
CTACCCCACCTTCCTCATTCTCTCTcgacagatttgttgcttaaatt


 


509
NM_025164 R
CTACCCCACCTTCCTCATTCTCTCTggcggtgggaaccttc


 


510
NM_001382 R
CTACCCCACCTTCCTCATTCTCTCTtaaagggcccgtacctctcc


 


511
NM_005422 R
CTACCCCACCTTCCTCATTCTCTCTtgccagagtaaacagaacacca


 


512
NM_003920 R
CTACCCCACCTTCCTCATTCTCTCTggaccggtccccg


 


513
NM_014770 R
CTACCCCACCTTCCTCATTCTCTCTaggtccgaggtgcaatcctaaa


 


514
NM_020366 R
CTACCCCACCTTCCTCATTCTCTCTgtaagagatcccagaggacact


 


515
NM_182932 R
CTACCCCACCTTCCTCATTCTCTCTccaggcagcaggcg


 


516
NM_016475 R
CTACCCCACCTTCCTCATTCTCTCTgcgggaccgtactcgt


 


517
NM_014994 R
CTACCCCACCTTCCTCATTCTCTCTatggtggcacgatcggc


 


518
NM_000499 R
CTACCCCACCTTCCTCATTCTCTCTccatcctggggcgc


 


519
NM_014699 R
CTACCCCACCTTCCTCATTCTCTCTtgagcatggcctttttgtcctc


 


520
NM_014906 R
CTACCCCACCTTCCTCATTCTCTCTcagcccacgctgccta


 


521
NM_014772 R
CTACCCCACCTTCCTCATTCTCTCTgccaagacagcccagtctag


 


522
NM_003259 R
CTACCCCACCTTCCTCATTCTCTCTggcaggagtgagcgac


 


523
NM_018837 R
CTACCCCACCTTCCTCATTCTCTCTggagggagccaaatgttcc


 


524
NM_002473 R
CTACCCCACCTTCCTCATTCTCTCTcggctcctcgccg


 


525
NM_005095 R
CTACCCCACCTTCCTCATTCTCTCTtctgagatcccacgggtcc


 


526
NM_004326 R
CTACCCCACCTTCCTCATTCTCTCTagttgctgctgcactggtg


 


527
NM_022093 R
CTACCCCACCTTCCTCATTCTCTCTcttctgacttccctcctccttc


 


528
NM_183361 R
CTACCCCACCTTCCTCATTCTCTCTggctccatccaggcttct


 


529
NM_024923 R
CTACCCCACCTTCCTCATTCTCTCTgagggagaaggcttgggg


 


530
NM_015136 R
CTACCCCACCTTCCTCATTCTCTCTcacccccacaggaaccc


 


531
NM_022131 R
CTACCCCACCTTCCTCATTCTCTCTcgccggcagcagc


 


532
NM_006218 R
CTACCCCACCTTCCTCATTCTCTCTgaggaggggcagagcc


 


533
NM_018986 R
CTACCCCACCTTCCTCATTCTCTCTggacggagcaggcag


 


534
NM_033632 R
CTACCCCACCTTCCTCATTCTCTCTtggttggggccccg


 


535
NM_001884 R
CTACCCCACCTTCCTCATTCTCTCTctgtgcccagaccttgtaaag


 


536
NM_000038 R
CTACCCCACCTTCCTCATTCTCTCTgcttctctctccgcttccc


 


537
NM_014005 R
CTACCCCACCTTCCTCATTCTCTCTatgcttgagattcttttcctga


 


538
NM_005322 R
CTACCCCACCTTCCTCATTCTCTCTtttcataagaatccattgggct


 


539
NM_019842 R
CTACCCCACCTTCCTCATTCTCTCTtcgaattctaaatccggacctg


 


540
NM_021807 R
CTACCCCACCTTCCTCATTCTCTCTtttttcagtttccttgctttta


 


541
NM_000598 R
CTACCCCACCTTCCTCATTCTCTCTcgagactcgcccggg


 


542
NM_004445 R
CTACCCCACCTTCCTCATTCTCTCTcctgcctgggctcg


 


543
NM_015225 R
CTACCCCACCTTCCTCATTCTCTCTgctgcaaccatggacagc


 


544
NM_152896 R
CTACCCCACCTTCCTCATTCTCTCTgagggggcgggtg


 


545
NM_018702 R
CTACCCCACCTTCCTCATTCTCTCTcgccctgctcagaaagaca


 


546
NM_020752 R
CTACCCCACCTTCCTCATTCTCTCTgctgctgctgctgc


 


547
NM_000314 R
CTACCCCACCTTCCTCATTCTCTCTgagatgggtgcgttgagc


 


548
NM_016234 R
CTACCCCACCTTCCTCATTCTCTCTgcctgccttggtctctgaa


 


549
NM_020975 R
CTACCCCACCTTCCTCATTCTCTCTcagtgcgggacgcg


 


550
NM_000124 R
CTACCCCACCTTCCTCATTCTCTCTcaaccatagacaccgccc


 


551
NM_000855 R
CTACCCCACCTTCCTCATTCTCTCTcgggtcggactgaggg


 


552
NM_020693 R
CTACCCCACCTTCCTCATTCTCTCTgcccttccaacccctc


 


553
NM_018400 R
CTACCCCACCTTCCTCATTCTCTCTctttcaggcaatgatgtcatct


 


554
NM_024546 R
CTACCCCACCTTCCTCATTCTCTCTgcaagattcctgcgaatgtgta


 


555
NM_003366 R
CTACCCCACCTTCCTCATTCTCTCTccgtgaaacaggggcct


 


556
NM_015202 R
CTACCCCACCTTCCTCATTCTCTCTccacttactgagcccgc


 


557
NM_000512 R
CTACCCCACCTTCCTCATTCTCTCTgtgtgcggatggggc


 


558
NM_002208 R
CTACCCCACCTTCCTCATTCTCTCTtccagcccagggtcctc


 


559
NM_000267 R
CTACCCCACCTTCCTCATTCTCTCTagagattgagagcgcggct


 


560
NM_005559 R
CTACCCCACCTTCCTCATTCTCTCTtggcctctgggtccc


 


561
NM_005359 R
CTACCCCACCTTCCTCATTCTCTCTttcctttctcccggctgc


 


562
NM_003245 R
CTACCCCACCTTCCTCATTCTCTCTtggggagaagggggcag


 


563
NM_012072 R
CTACCCCACCTTCCTCATTCTCTCTctgccgggtccctgg


 


564
NM_006275 R
CTACCCCACCTTCCTCATTCTCTCTcgggaggcgggct


 


565
NM_003253 R
CTACCCCACCTTCCTCATTCTCTCTccgattgggccgcc


 


566
NM_003906 R
CTACCCCACCTTCCTCATTCTCTCTatgttctgctacaagtctaaga


 


567
NM_006932 R
CTACCCCACCTTCCTCATTCTCTCTgcccgtccagccg


 


568
NM_004985 R
CTACCCCACCTTCCTCATTCTCTCTATCGATGCGTTCCGCG


 


569
NM_007355 R
CTACCCCACCTTCCTCATTCTCTCTactgcgtgccccaagtc


 


570
AK311497 R
CTACCCCACCTTCCTCATTCTCTCTgcgggtccctggg


 


571
NM_004387 R
CTACCCCACCTTCCTCATTCTCTCTAAGACACCAGGCTGCAGGAT


 


572
NM_006941_1 R
CTACCCCACCTTCCTCATTCTCTCTTCCTGCGCGCTGC


 


573
NM_005559 R
CTACCCCACCTTCCTCATTCTCTCTtggcctctgggtccc


 


574
NM_006218 R
CTACCCCACCTTCCTCATTCTCTCTgaggaggggcagagcc


 


575
NM_003105 R
CTACCCCACCTTCCTCATTCTCTCTCCTAGAACGCAACCAACAAGA


 


576
NM_000546 R
CTACCCCACCTTCCTCATTCTCTCTGGACAGTCGCCATGACAA







Universal Primers









577
U1
GGT GTG TGT GGG TAG TTG GTG AT


 


578
U2
/5Phos/AGA GAA TGA GGA AGG TGG GGT AG/3SpC3/


 


579
U2′
CTA CCC CAC CTT CCT CAT TCT CT


 


580
454A: Sample
GCC TCC CTC GCG CCA TCA G (5bp barcode) GGT GTG



Specific
TGT GGG TAG TTG GTG AT



Barcode: U1



 


581
454B: Sample
GCC TTG CCA GCC CGC TCA G (5bp barcode) CTA CCC



Specific
CAC CTT CCT CAT TCT CT



Barcode: U2′









Bisulfite Patch PCR

Genomic DNA from cancer and adjacent normal tissue was obtained from Biochain (www.biochain.com) for both breast (catalog number D8235086) and colon (catalog number 8235090). Patient information and lot numbers are listed in TABLE F. Each patient sample was aliquoted into a well of a 96-well plate and digested with the AluI restriction endonuclease in 10 ul total volume reaction containing 250 ng DNA, 10 units (U) AluI enzyme (NEB), and 1× NEBuffer 2 (NEB). This reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step.
Patch driven ligation of the universal primers to selected fragments was performed by addition of more reactants to the initial tube to result in the following final concentrations: 2 nM each Patch oligo, 200 nM U1 primer, 200 nM U2 primer (contains 5′ phosphate and 3′ three carbon spacer), 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.
Incorrect products, template genomic DNA and excess primer were degraded by the direct addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes, and held at 4° C.
The reactions were then treated with sodium bisulfite to convert unmethylated cytosines to uracil. This was achieved by using the EZ DNA Methylation Gold Bisulfite Treatment Kit (Zymo Research) following the manufacturer's instructions, with one exception. Since the sample volume after the exonuclease treatment is 27 ul, the CT Conversion Reagent from the kit is made by adding 830 ul dH2O instead of 900 ul dH2O. The DNA is eluted from the columns in the final step with 10 ul M-Elution buffer.
The universal primers are then used to PCR amplify the selected bisulfite converted loci from each sample. A different pair of universal primers is used to PCR amplify each sample, and they are distinguished by a five base-pair sample-specific DNA barcode that resides between the universal primer sequence and the 454 machine specific sequence (TABLE J). There are 1,024 possible 5 bp DNA sequences, and we selected 48 sample-specific barcodes, one for each sample, that did not contain homopolymers and had the least sequence similarity to each other (The barcodes used for each patient are listed in TABLE F). For the PCR we added reagents to the last 10 ul column elution to result in these final concentrations in 50 ul: 0.5 uM each Barcoded U1, 0.5 uM each Barcoded U2′, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl2, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 35 cycles, and held at 4° C. The PCR product smear between the expected sizes was confirmed by running 20 ul of the PCR product from each sample on a 3% Metaphor Agarose gel (Lonza). We then pooled 5 ul from each sample into a single tube and purified this pool on a Qiaquick Spin Column (Qiagen). The eluted DNA was quantified on the Nanodrop (www.nanodrop.com) as well as on a plate reader (BioTek Synergy HT) using PicoGreen (Invitrogen) following the manufacturer's instructions. This pooled sample was then prepared and sequenced on the 454 Life Sciences/Roche FLX machine following the manufacturer's instructions.
Sequencing Data Analysis
We obtained 97,115 sequencing reads. To determine which sequences matched our targets, we aligned the reads against a database of reference sequences for each target using WU-BLASTN (http://blast.wustl.edu). Since the sequences are sodium bisulfite treated, we substituted a T in place of C in the genomic sequence at non-CpG positions in the reference sequences. We then determined how many reads matched significantly to each promoter (BLAST smallest sum probability (P)<0.001), and put all reads from each promoter in a separate file. We computed the correlation between the number of reads and the amplicon length for each promoter using linear regression. We identified which sample each read came from by matching the first five bases of the read to the list of sample-specific barcode and corresponding patients. To determine the reproducibility of the method, we computed number of reads for each locus in each sample, and calculated the squared correlation coefficient (R2) between two samples for all possible pairs of samples. The mean of these correlation coefficients represents the average correlation between the number of reads per locus across samples. For each promoter, we used CLUSTALW to generate a multiple sequence alignment of all of the reads and the reference sequence (Larkin et al. 2007). We identified germline SNPs in the sequences by looking for variants in the reads and comparing these to known SNPs reported on the UCSC Genome Browser (www.genome.ucsc.edu). To visualize these multiple sequence alignments we create one matrix per promoter where the first column identifies the sample from which the read originated (1-48), and the remaining columns are coded for the base in the read, where C's are replaced with 8, the two alleles at SNP positions are replaced with 5 and 12, and the remaining bases are converted to 0. This matrix was then visualized as an image using the Matlab software package (The Mathworks). The matrix was sorted by sample type (the first column) and further calculations regarding the amount of methylation per read and per sample were computed using Matlab (The MathWorks Inc.).
To quantify the sensitivity and specificity of each locus exhibiting tumor-specific methylation we used a threshold to classify a locus as methylated or unmethylated in each sample. We queried many CpGs for each locus with the bisulfite sequencing. We used this information to find the optimal classifier of DNA methylation to distinguish tumor and normal samples. We search across all possible values for two parameters: % of CpGs per molecule and % of reads per sample. We found that the optimal classifier between tumor and normal was to classify a sample as ‘methylated’ if more than 20% of CpG positions per molecule were methylated in more than 35% of molecules. The fraction of samples that were classified as methylated is listed in TABLE I for each locus.
REFERENCES FOR EXAMPLE 7


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Example 8: Nucleic Acid Patch PCR with Ends Defined by Oligonucleotide-Directed FokI Digestion
This example details creating defined ends of a nucleic acid sequence by using oligonucleotide-directed digestion on nucleic acid templates. The method is depicted in FIG. 13.
Template Preparation
FokI-directing DNA oligonucleotides were designed to anneal upstream and downstream of each of 96 targeted exons in the human genome. These loci were selected because they are genes implicated in pediatric acute lymphoblastic leukemia. The oligos contained the recognition sequence of the FokI restriction endonuclease. Human genomic DNA from the blood of healthy individuals (Promega) was incubated with FokI-directing oligonucleotides in a reaction containing appropriate buffer for the FokI enzyme, NEBuffer3 (NEB) and a final concentration of 0.1% Tween80 (Sigma) in a total volume of 9 ul. This mixture was denatured at 98° C. for 15 minutes and held at 37° C. for 5 minutes. FokI enzyme (NEB) was then added to the reaction so that there was 4 U of enzyme in a 10 ul reaction. The reaction was incubated at 37° C. for 1 hour, followed by heat inactivation of the enzyme at 65° C. for 20 min, and held at 4° C. until the subsequent step. Control reactions lacking Tween80, FokI-directing oligonucleotides, FokI enzyme, or genomic DNA were also performed.
Nucleic Acid Patch Ligation
Nucleic acid patch oligos were designed as described in Example 2 but were designed to anneal adjacent to the FokI-digested cut sites upstream and downstream of a targeted 96 exons in the human genome. Nucleic Acid Patch driven ligation of the universal primers to selected fragments was performed essentially as in Example 2. Briefly, the following reactants were added to the FokI digest to result in the following final concentrations: 2 nM each Nucleic Acid Patch oligo, 200 nM Universal Primer 1, 200 nM Universal Primer 2 with 5′ phosphate and 3′ three carbon spacer, 5 U Ampligase (Epicentre), and 1× Ampligase Reaction Buffer (Epicentre) in a total volume of 25 ul. This reaction was incubated at 95° C. for 15 minutes followed by (94° C. for 30 sec, 65° C. for 8 min) for 100 cycles, and held at 4° C.
Incorrect products, template genomic DNA and excess primer were degraded by the direct addition of 10 U Exonuclease I (USB) and 200 U Exonuclease III (Epicentre) to the reaction. This mix was incubated at 37° C. for 1 hour followed by heat inactivation at 95° C. for 20 minutes and then held at 4° C.
PCR Amplification
The universal primers were then used to PCR amplify the selected loci from each sample. For the PCR, reagents were added to the reactions to result in these final concentrations in 50 ul: 0.5 uM each Universal Primer, 10 U Platinum Taq Polymerase (Invitrogen), 0.5 mM each dNTP, 2 mM MgCl2, 0.5M Betaine, 20 mM Tris-HCl pH 8.4 and 50 mM KCl. This reaction was incubated at 93° C. for 2 minutes followed by (93° C. for 30 sec, 57° C. for 6 min) for 35 cycles, and held at 4° C. An aliquot of the reactions was analyzed by gel elctrophoresis on a 2% agarose gel (Lonza).
Results
Defining template ends using oligo-directed FokI digestion was successful (FIG. 14). A smear of PCR products of the expected sizes was detected on the agarose gel.

## Claims

1. A method of amplifying at least two different nucleic acid sequences, the method comprising:
a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by:
i) annealing oligonucleotides to the at least two nucleic acid sequences, wherein the oligonucleotides comprise a locus specific sequence and form a hairpin structure comprising a Type IIS restriction enzyme recognition sequence, and
ii) contacting the at least two nucleic acid sequences with a Type IIS restriction enzyme, under conditions suitable for the enzymes to cleave the at least two nucleic acid sequences creating known 5′ and 3′ ends,

b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of known ends from step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and
d) amplifying the nucleic acid sequences of step (c).

2. The method of claim 1, wherein each nucleic acid sequence is encoded by genomic DNA.

3. The method of claim 1, wherein the downstream universal primer comprises a protecting group.

4. The method of claim 1, further comprising sequencing the products of step (d).

5. The method of claim 4, wherein the amplifying of step (d) is performed with an upstream and a downstream amplification primer, wherein the upstream amplification primer comprises a first universal sequence and a sequence corresponding to the upstream universal primer of step (b), and wherein the downstream amplification primer comprises a second universal sequence and a sequence corresponding to the downstream universal primer of step (b), wherein the products of step (d) are sequenced using an upstream and a downstream sequencing primer corresponding to the first and second universal sequences, respectively.

6. The method of claim 1, wherein step (c) additionally includes treating the ligated nucleic acid sequences with bisulfite, wherein the amplifying of step (d) amplifies the bisulfite-treated nucleic acid sequences.

7. The method of claim 1, wherein the oligonucleotides comprise a locus specific sequence comprising 10 or more bases.

8. A method of amplifying at least two different nucleic acid sequences, the method comprising:
a) creating known 5′ and 3′ ends of at least two nucleic acid sequences by:
i) annealing oligonucleotides to the at least two nucleic acid sequences, wherein the oligonucleotides comprise a locus specific sequence comprising 10 or more bases and a Type IIS restriction enzyme recognition sequence, and
ii) contacting the at least two nucleic acid sequences with a Type IIS restriction enzyme, under conditions suitable for the enzymes to cleave the at least two nucleic acid sequences creating known 5′ and 3′ ends,

b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of known ends from step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence; and
d) amplifying the nucleic acid sequences of step (c).

9. The method of claim 8, wherein each nucleic acid sequence is encoded by genomic DNA.

10. The method of claim 8, wherein the downstream universal primer comprises a protecting group.

11. The method of claim 8, further comprising sequencing the products of step (d).

12. The method of claim 11, wherein the amplifying of step (d) is performed with an upstream and a downstream amplification primer, wherein the upstream amplification primer comprises a first universal sequence and a sequence corresponding to the upstream universal primer of step (b), and wherein the downstream amplification primer comprises a second universal sequence and a sequence corresponding to the downstream universal primer of step (b), wherein the products of step (d) are sequenced using an upstream and a downstream sequencing primer corresponding to the first and second universal sequences, respectively.

13. The method of claim 8, wherein step (c) additionally includes treating the ligated nucleic acid sequences with bisulfite, wherein the amplifying of step (d) amplifies the bisulfite-treated nucleic acid sequences.

14. The method of claim 8, wherein the oligonucleotides form a hairpin structure comprising the Type IIS restriction enzyme recognition sequence.

15. A method of detecting short tandem repeats, the method comprising:
a) creating known 5′ and 3′ ends of at least two nucleic acid sequences, wherein the known 5′ and 3′ ends are immediately adjacent to a short tandem repeat, by:
i) annealing oligonucleotides to the at least two nucleic acid sequences, wherein the oligonucleotides comprise a locus specific sequence and a Type IIS restriction enzyme recognition sequence, and
ii) contacting the at least two nucleic acid sequences with a Type IIS restriction enzyme, under conditions suitable for the enzymes to cleave the at least two nucleic acid sequences creating known 5′ and 3′ ends,

b) annealing an upstream and a downstream nucleic acid patch to each nucleic acid sequence of known ends from step (a), and annealing an upstream universal primer to the upstream patch, and a downstream universal primer to the downstream patch;
c) ligating the upstream universal primer and the downstream universal primer to each nucleic acid sequence;
d) amplifying the nucleic acid sequences of step (c); and
e) sequencing the products of step (d) to detect the length of the short tandem repeat.

16. The method of claim 15, wherein each nucleic acid sequence is encoded by genomic DNA.

17. The method of claim 15, wherein the downstream universal primer comprises a protecting group.

18. The method of claim 15, wherein the amplifying of step (d) is performed with an upstream and a downstream amplification primer, wherein the upstream amplification primer comprises a first universal sequence and a sequence corresponding to the upstream universal primer of step (b), and wherein the downstream amplification primer comprises a second universal sequence and a sequence corresponding to the downstream universal primer of step (b), wherein the products of step (d) are sequenced using an upstream and a downstream sequencing primer corresponding to the first and second universal sequences, respectively.

19. The method of claim 15, wherein step (c) additionally includes treating the ligated nucleic acid sequences with bisulfite, wherein the amplifying of step (d) amplifies the bisulfite-treated nucleic acid sequences.

20. The method of claim 15, wherein the oligonucleotides form a hairpin structure comprising the Type IIS restriction enzyme recognition sequence.

21. The method of claim 15, wherein the oligonucleotides comprise a locus specific sequence comprising 10 or more bases.