DIGITAL SEQUENCE ANALYSIS OF DNA METHYLATION
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.
1 - 30 . (canceled)
31 . A method for identifying a cancer-associated locus, comprising:
a) identifying one or more cancer-associated loci in nucleic acid obtained from a sample from a subject by comparing nucleic acid sequence from a cancer cell from said subject to nucleic acid from a normal cell;
b) generating an assay to detect said one or more cancer-associated loci identified in step a); and
c) conducting said assay, wherein said assay comprises:
i) preparing a library of nucleic acid suspected of containing said one or more cancer-associated loci;
ii) amplifying nucleic acid in said library; and
iii) sequencing said library to identify sequences associated with said one or more cancer-associated loci;
wherein said conducting comprises enriching said sample for nucleic acid comprising said one or more cancer-associated loci, if present; adding index tags to said nucleic acid; and sequencing amplicons over 1000 times.
32 . The method of claim 31 , wherein said conducting comprises sequencing amplicons over 100,000 times.
33 . The method of claim 31 , wherein said one or more cancer-associated loci are in one or more marker nucleic acids selected from bmp-3, bmp-4, SFRP2, vimentin, septin9, ALX4, EYA4, TFPI2, NDRG4, FOXE1, long DNA, BAT-26, K-ras, APC, melanoma antigen gene, p53, BRAF, and PIK3CA nucleic acids.
34 . The method of claim 31 , wherein said one or more cancer-associated loci comprise CpG dinucleotide loci.
35 . The method of claim 31 , wherein said one or more cancer-associated loci are differentially methylated in cancer samples compared to normal samples.
36 . The method of claim 31 , wherein said enriching comprises amplification of said nucleic acid with one or more primers that selectively amplify a cancer-associated locus.
37 . The method of claim 31 , wherein said cancer-associated loci are associated with colorectal cancer or colorectal adenoma.
38 . The method of claim 31 , wherein said amplifying comprises PCR.
39 . The method of claim 38 , wherein said PCR comprises digital PCR.
40 . The method of claim 38 , wherein said PCR comprises nested PCR.