Methods and compositions for generating and amplifying dna libraries for sensitive detection and analysis of dna methylation

Abstract

The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules

1 - 73 . (canceled) 
     
     
         74 . A method for preparing a DNA molecule, the method comprising:
 a) providing a sample comprising methylated nucleic acids and non-methylated nucleic acids;   b) generating a nucleic acid library from the methylated nucleic acids and the non-methylated nucleic acids by attaching hairpin adaptors;   c) digesting the nucleic acid library with a methylation-sensitive restriction enzyme; and   b) detecting methylation sites within nucleic acids of the digested nucleic acid library;   wherein the detecting comprises detecting methylated nucleic acids that comprise less than 10% of the nucleic acids in the sample.   
     
     
         75 . The method of  claim 74 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 1% of the nucleic acids in the sample. 
     
     
         76 . The method of  claim 74 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 0.1° A of the nucleic acids in the sample. 
     
     
         77 . The method of  claim 74 , wherein the detecting comprises detecting methylated nucleic acids that comprise from 10% to 0.01% of the nucleic acids in the sample. 
     
     
         78 . The method of  claim 74 , wherein the nucleic acids are from serum DNA. 
     
     
         79 . The method of  claim 74 , wherein the nucleic acids are from circulating cell-free DNA. 
     
     
         80 . The method of  claim 74 , wherein the nucleic acids are from an apoptosed cell. 
     
     
         81 . The method of  claim 74 , wherein the nucleic acid are from a source selected from the group consisting of: biopsy materials, pap smears, serum, and plasma, and any combinations thereof. 
     
     
         82 . The method of  claim 74 , wherein the attaching hairpin adaptors comprises ligating. 
     
     
         83 . The method of  claim 82 , wherein the ligating comprises ligating a first adaptor comprising a known sequence and a nonblocked 3′ end to an end of the digested fragments to produce an adaptor-linked molecule, wherein the 5′ end of the digested fragments are attached to the nonblocked 3′ end of the first adaptor, leaving a nick site between a juxtaposed 3′ end of the digested fragments and a 5′ end of the first adaptor. 
     
     
         84 . The method of  claim 83 , further comprising extending the juxtaposed 3′ end of the digested fragments from the nick site by polymerization. 
     
     
         85 . The method of  claim 74 , wherein the detecting comprises using a detection method selected from the group consisting of: sequencing, quantitative real-time PCR, ligation chain reaction, ligation-mediated PCR, probe hybridization, probe amplification, and microarray hybridization, and any combinations thereof. 
     
     
         86 . The method of  claim 74 , wherein the generating comprises generating a library from 1 to 100 ng of said digested fragments. 
     
     
         87 . The method of  claim 74 , wherein the method is performed in a single tube. 
     
     
         88 . The method of  claim 74 , wherein the methylation sensitive restriction enzyme comprises one or more methylation sensitive restriction endonucleases selected from the group consisting of: McrBC, Aci I, Bst UI, Hha I, HinP1, Hpa II, Hpy 991, Ava I, Bce AI, Bsa HI, Bsi E1, and Hga I, and any combinations thereof. 
     
     
         89 . A method for preparing a DNA molecule, the method comprising:
 a) providing a sample comprising methylated nucleic acids and non-methylated nucleic acids;   b) digesting the methylated and non-methylated nucleic acids with a methylation-sensitive restriction enzyme, thereby creating digested fragments;   c) generating a library from the digested fragments; and   d) detecting methylation sites within the library;   wherein said detecting comprises detecting methylated nucleic acids that comprise less than 10% of the nucleic acids in said sample.   
     
     
         90 . The method of  claim 89 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 1% of the nucleic acids in the sample. 
     
     
         91 . The method of  claim 89 , wherein the detecting comprises detecting methylated nucleic acids that comprise less than 0.1° A of the nucleic acids in the sample. 
     
     
         92 . The method of  claim 89 , wherein the detecting comprises detecting methylated nucleic acids that comprise from 10% to 0.01% of the nucleic acids in the sample. 
     
     
         93 . The method of  claim 89 , wherein said nucleic acids are from serum DNA. 
     
     
         94 . The method of  claim 89 , wherein the nucleic acids are from circulating cell-free DNA. 
     
     
         95 . The method of  claim 89 , wherein the nucleic acids are from an apoptosed cell. 
     
     
         96 . The method of  claim 89 , wherein the nucleic acids are from a source from the group consisting of: biopsy materials, pap smears, serum, and plasma, and any combinations thereof. 
     
     
         97 . The method of  claim 89 , wherein the generating of the library comprises incorporating a nucleic acid molecule into at least some of the digested fragments to provide first modified DNA molecules, by incorporating at least one primer from a plurality of primers, said primers comprising a 5′ constant sequence and a 3′ variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein the sequence of the constant and variable regions consists essentially of only two types of non-complementary nucleotides selected from the group consisting of adenines and guanines; adenines and cytosines; guanines and thymidines; and cytosines and thymidines, such that the primers of the population will not cross-hybridize or self-hybridize under amplification conditions. 
     
     
         98 . The method of  claim 97 , further comprising amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules. 
     
     
         99 . The method of  claim 89 , wherein the generating of the library comprises ligating hairpin adaptors to the digested fragments. 
     
     
         100 . The method of  claim 89 , wherein the detecting comprises using a detection method selected from the group consisting of: sequencing, quantitative real-time PCR, ligation chain reaction, ligation-mediated PCR, probe hybridization, probe amplification, and microarray hybridization, and any combinations thereof. 
     
     
         101 . The method of  claim 89 , wherein the generating comprises generating a library from 1 to 100 ng of the digested fragments. 
     
     
         102 . The method of  claim 89 , wherein the method is performed in a single tube. 
     
     
         103 . The method of  claim 89 , wherein the methylation sensitive restriction enzyme comprises one or more methylation sensitive restriction endonucleases selected from the group consisting of: McrBC, Aci I, Bst UI, Hha I, HinP1, Hpa II, Hpy 991, Ava I, Bce AI, Bsa HI, Bsi E1, and Hga I, and any combinations thereof.