Digital sequence analysis of DNA methylation
AssigneesExact Sciences Corporation, Mayo Foundation for Medical Research and Education
InventorsDavid A. Ahlquist, William R. Taylor, Hongzhi Zou, Graham P. Lidgard
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Abstract
The present invention relates to methods and compositions for determination of and uses of specific methylation patterns indicative of adenoma and carcinoma. In particular, the invention relates to analysis of defined CpG loci that are coordinately methylated in DNAs from cancer and adenoma samples, methods for identifying coordinately methylated loci, and methods of using analysis of coordinately methylated loci in one or more marker regions in the design of assays for adenoma and cancer.
Claims19
A method of making an assay for detecting DNA amplified from a methylated marker DNA in the presence of DNA amplified from the unmethylated marker DNA that exhibits background methylation, the method comprising:
- a) isolating marker DNA from a first group of samples in which the marker DNA is unmethylated DNA exhibiting background methylation and from a second group of samples in which the marker DNA is methylated;
- b) processing the isolated marker DNA using digital sequencing and/or digital polymerase chain reaction (PCR) to determine the methylation status for three or more CpG loci in each of at least 1000 individual copies of the isolated marker DNA from each of the first and second groups of samples;
- c) for each of the three or more CpG loci, determining a ratio between a mean methylation at that CpG locus in the first group of samples to a mean methylation at the corresponding CpG locus in the second group of samples;
- d) selecting a subset of at least three CpG loci for which the percentage of individual copies of the marker DNA from the first group of samples that are methylated at all of the at least three CpG loci in the subset is less than the percentage of individual copies of the marker DNA from the second group of samples that are methylated at all of the at least three CpG loci in the subset;
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- e) creating a detection assay that detects the methylation status of all of the at least three CpG loci in the subset of CpG loci in a strand of the marker DNA, wherein the absence of methylation at any one of the CpG loci in the subset of CpG loci in a strand of the marker DNA produces an assay result classifying that strand of the marker DNA as not methylated,
- wherein creating the detection assay comprises synthesizing at least one oligonucleotide complementary to a segment of nucleic acid comprising the subset of CpG loci.
A method of making an assay for characterizing DNA amplified from a methylated marker DNA in the presence of DNA amplified from the unmethylated marker DNA that exhibits background methylation, the method comprising:
- a) isolating marker DNA from a first group of samples in which the marker DNA is unmethylated DNA exhibiting background methylation and from a second group of samples in which the marker DNA is methylated;
- b) treating the isolated marker DNA under conditions wherein unmethylated cytosine (C) bases are converted to uracil (U) bases to produce treated isolated marker DNA;
- c) processing the treated isolated marker DNA using digital sequencing and/or digital polymerase chain reaction (PCR) to detect unconverted C bases in three or more CpG loci in each of at least 1000 individual copies of the treated isolated marker DNA from each of the first and second groups of samples;
- d) for each of the three or more CpG loci, determining a ratio between a mean unconverted C in that CpG locus in the first group of samples to a mean unconverted C in the corresponding CpG locus in the second group of samples;
- e) selecting a subset of at least three CpG loci for which the percentage of individual copies of the treated isolated marker DNA from the first group of samples that have unconverted C in all of the at least three CpG loci in the subset is less than the percentage of individual copies of the marker DNA from the second group of samples that have unconverted C in all of the at least three CpG loci in the subset;
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- f) creating a detection assay that detects the presence or absence of unconverted C in all of the at least three CpG loci in the subset of CpG loci in a strand of treated marker DNA, wherein the absence of unconverted C in any one of the CpG loci in the subset of CpG loci in a strand of the marker DNA produces an assay result classifying that strand of the marker DNA as not methylated,
- wherein creating the detection assay comprises synthesizing at least one oligonucleotide complementary to a segment of nucleic acid comprising the subset of CpG loci.
Dependencies are inferred from the claim text.