Methods for identifying multiple dna alteration markers in a large background of wild-type dna

Abstract

Methods for simultaneously surveying the status of a large number of DNA mutation markers are described. In addition, methods for simultaneously determining the methylation status at multiple sites of a collection of genes, in a single assay, are described.

1 . A method for surveying the methylation status of a large number of CpG sites in a large background of unmethylated DNA, the method comprising:
 providing a sample including methylated DNA and unmethylated DNA;   treating the sample to convert cytosine groups in the unmethylated DNA to uracil groups;   amplifying the treated sample by a first PCR with primers to thereby yield DNA fragments in which all uracil groups are converted to thymine groups;   enriching the methylated DNA by performing one or multiple cycles of methylation-specific enrichment cycles to thereby form an enriched system;   amplifying the enriched system by a second PCR to generate sufficient amounts of methylated DNA for detection; and   surveying the methylation status of the CpG sites.   
     
     
         2 . The method of  claim 1  wherein the sample is obtained from clinical biospecimens collected from patients, the biospecimens selected from the group consisting of human tumor tissues, peripheral blood, stool, urine, bodily fluids, washing fluids associated with medical procedures, and combinations thereof. 
     
     
         3 . The method of  claim 1  wherein the molar ratio of the unmethylated DNA to the methylated DNA is from about 2:1 to about 100,000:1 in the sample. 
     
     
         4 . The method of  claim 1  wherein the CpG sites are in one or more genes. 
     
     
         5 . The method of  claim 1  wherein the treating is performed by using a bisulfite reaction. 
     
     
         6 . The method of  claim 1  wherein the first PCR is multiplexed PCR utilizing two or more pairs of PCR primers, wherein each pair of primers includes a forward primer and a reverse primer, wherein each pair of primers is adapted to amplify a sequence containing one or more of the CpG sites and wherein each primer further includes both a target-specific part and a universal tail part, wherein the target specific part flanks the sequence to allow for amplification of the sequence and the sequence of the universal tail part is the same for all forward and reverse primers, wherein the universal tail does not bind any DNA fragments produced by the treating operation. 
     
     
         7 . The method of  claim 1  wherein the methylation-specific enrichment cycles are selected from the group consisting of (i) depleting unmethylated DNA, (ii) selectively capturing methylated DNA, and (iii) a combination of (i) and (ii). 
     
     
         8 . The method of  claim 1  wherein enrichment of the methylated DNA fragments containing the methylated cytosine at CpG sites from the amplicons of the multiplexed PCR is performed by at least one of (i) methylation-specific hybridization and extraction and (ii) competing mutation-specific hybridization and extraction. 
     
     
         9 . The method of  claim 8  wherein enriching is performed by methylation-specific hybridization and extraction, wherein a plurality of methylation-specific probes are contacted with the DNA fragments of the first PCR under hybridization conditions wherein each methylation-specific probe preferentially forms hybrids with a methylated sequence, wherein the methylation-specific probes are further attached to a first binding molecule that is capable of binding to a second binding molecule that is attached to a solid support, and wherein after hybridization, the hybrids are captured by the solid support containing the second binding molecule. 
     
     
         10 . The method of  claim 9  wherein the number of methylation-specific hybridization and extraction cycles performed ranges from one cycle to five cycles. 
     
     
         11 . The method of  claim 10  wherein the number of cycles is one. 
     
     
         12 . The method of  claim 10  wherein the number of cycles is two or more. 
     
     
         13 . The method of  claim 12  wherein the methylation-specific hybridization and extraction is repeated, wherein the DNA fragments captured by the solid support are released from the solid support and further subjected to additional cycles of the methylation-specific hybridization and extraction. 
     
     
         14 . The method of  claim 9  wherein the methylation-specific probes are selected from the group consisting of oligonucleotides, peptide nucleic acids, locked nucleic acids, and combinations thereof. 
     
     
         15 . The method of  claim 9  wherein the first binding molecule is selected from the group consisting of biotin, streptavidin, and combinations thereof and the second binding molecule is selected from the group consisting of streptavidin, biotin, and combinations thereof. 
     
     
         16 . The method of  claim 8  wherein enriching of the methylated DNA fragments is performed by competing methylation-specific hybridization and extraction, wherein a plurality of methylation-specific probes and unmethylated competitor probes are contacted with the DNA fragments of the first PCR under hybridization conditions wherein the methylation-specific probes preferentially form hybrids with methylated DNA fragments, the corresponding unmethylated competitor probes preferentially form hybrids with the corresponding unmethylated DNA fragments, wherein the methylation-specific probes are further attached to a first binding molecule that is capable of binding to a second binding molecule that is attached to a solid support, and wherein after hybridization, the hybrids are captured by the solid support containing the second binding molecule. 
     
     
         17 . The method of  claim 16  wherein the number of competing methylation-specific hybridization and extraction cycles performed ranges from one cycle to five cycles. 
     
     
         18 . The method of  claim 17  wherein the number of cycles is one. 
     
     
         19 . The method of  claim 17  wherein the number of cycles is two or more. 
     
     
         20 . The method of  claim 19  wherein the competing methylation-specific hybridization and extraction is repeated, wherein the DNA fragments captured by the solid support are released from the solid support and further subjected to additional cycles of the competing methylation-specific hybridization and extraction. 
     
     
         21 . The method of  claim 16  wherein the methylation-specific probes are selected from the group consisting of oligonucleotides, peptide nucleic acids, locked nucleic acids, and combinations thereof. 
     
     
         22 . The method of  claim 16  wherein the first binding molecule is selected from the group consisting of biotin, streptavidin, and combinations thereof and the second binding molecule is selected from the group consisting of streptavidin, biotin, and combinations thereof. 
     
     
         23 . The method of  claim 16  wherein the molar ratio of the methylation-specific hybridization probes to the corresponding unmethylated competitor probes is from about 0.02:1 to about 10:1. 
     
     
         24 . The method of  claim 1  wherein the first PCR uses two or more pairs of primers, each pair of primers includes a forward primer and a reverse primer, each pair of primers including a universal tail, the second PCR is performed by contacting the enriched methylated DNA fragments with universal primers, wherein the universal primers hybridize to the universal tails of the forward and reverse primers to amplify all enriched DNA fragments. 
     
     
         25 . The method of  claim 24  wherein the second PCR utilizes one universal primer when the universal tail of the forward and reverse primers of the first PCR is same, and wherein the second PCR utilizes two universal primers when the universal tail of the forward and reverse primers of the first PCR are different, wherein the one universal primer matches to the universal tail of the forward primers of the first PCR while the other universal primer matches to the universal tail of the reverse primers of the first PCR. 
     
     
         26 . A method for producing sufficiently pure methylated DNA fragments for determining the methylation status at a plurality of DNA CpG sites in a large background of unmethylated DNA, the method comprising: providing a DNA sample containing both methylated and unmethylated DNA; subjecting the DNA sample to bisulfite treatment; amplifying DNA sequences containing methylated CpG sites by multiplexed PCR to thereby produce amplicons; and enriching methylated DNA fragments containing the methylated CpG sites from the amplicons of the PCR. 
     
     
         27 . The method of  claim 26  wherein the DNA sample is obtained from clinical biospecimens collected from patients, the biospecimens selected from the group consisting of human tumor tissues, peripheral blood, stool, urine, bodily fluids, washing fluids associated with medical procedures, and combinations thereof. 
     
     
         28 . The method of  claim 26  wherein the molar ratio of the unmethylated DNA to the methylated DNA is about 2:1 to about 100,000:1 in the DNA sample. 
     
     
         29 . The method of  claim 26  wherein the CpG sites are in one or more genes. 
     
     
         30 . The method of claim  72  wherein the bisulfite treatment converts unmethylated cytosine in the CpG sites to uracil whereas methylated cytosine in the CpG sites are unaltered. 
     
     
         31 . The method of  claim 30  wherein the multiplexed PCR utilizes two or more pairs of PCR primers, wherein each pair of primers hybridize to a sequence containing one or more of the CpG sites for amplification of the sequence. 
     
     
         32 . The method of  claim 31  wherein the multiplexed PCR converts uracil to thymine. 
     
     
         33 . The method of  claim 26  wherein enrichment of the methylated DNA fragments containing the methylated CpG sites from the amplicons of the multiplexed PCR is performed by at least one of (i) methylation-specific hybridization and extraction and (ii) competing methylation-specific hybridization and extraction. 
     
     
         34 . The method of  claim 33  wherein enriching is performed by methylation-specific hybridization and extraction, wherein a plurality of methylation-specific probes are contacted with the DNA fragments of the first PCR under hybridization conditions wherein each methylation-specific probe preferentially forms hybrids with a methylated sequence, wherein the methylation-specific probes are further attached to a first binding molecule that is capable of binding to a second binding molecule that is attached to a solid support, and wherein after hybridization, the hybrids are captured by the solid support containing the second binding molecule. 
     
     
         35 . The method of  claim 34  wherein the number of competing methylation-specific hybridization and extraction cycles performed ranges from one cycle to five cycles. 
     
     
         36 . The method of  claim 35  wherein the number of cycles is one. 
     
     
         37 . The method of  claim 35  wherein the number of cycles is two or more. 
     
     
         38 . The method of  claim 37  wherein the methylation-specific hybridization and extraction is repeated, wherein the DNA fragments captured by the solid support are released from the solid support and further subjected to additional cycles of the methylation-specific hybridization and extraction. 
     
     
         39 . The method of  claim 34  wherein the methylation-specific probes are selected from the group consisting of oligonucleotides, peptide nucleic acids, locked nucleic acids, and combinations thereof. 
     
     
         40 . The method of  claim 34  wherein the first binding molecule is selected from the group consisting of biotin, streptavidin, and combinations thereof and the second binding molecule is selected from the group consisting of streptavidin, biotin, and combinations thereof. 
     
     
         41 . The method of  claim 33  wherein enriching of the methylated DNA fragments is performed by competing methylation-specific hybridization and extraction, wherein a plurality of methylation-specific probes and unmethylated competitor probes are contacted with the DNA fragments of the first PCR under hybridization conditions wherein the methylation-specific probes preferentially form hybrids with methylated DNA fragments, the corresponding unmethylated competitor probes preferentially form hybrids with the corresponding unmethylated DNA fragments, wherein the methylation-specific probes are further attached to a first binding molecule that is capable of binding to a second binding molecule that is attached to a solid support, and wherein after hybridization, the hybrids are captured by the solid support containing the second binding molecule. 
     
     
         42 . The method of  claim 41  wherein the number of competing methylation-specific hybridization and extraction cycles performed ranges from one cycle to five cycles. 
     
     
         43 . The method of  claim 42  wherein the number of cycles is one. 
     
     
         44 . The method of  claim 42  wherein the number of cycles is two or more. 
     
     
         45 . The method of  claim 44  wherein the competing methylation-specific hybridization and extraction is repeated, wherein the DNA fragments captured by the solid support are released from the solid support and further subjected to additional cycles of the competing methylation-specific hybridization and extraction. 
     
     
         46 . The method of  claim 41  wherein the methylation-specific probes are selected from the group consisting of oligonucleotides, peptide nucleic acids, locked nucleic acids, and combinations thereof. 
     
     
         47 . The method of  claim 41  wherein the first binding molecule is selected from the group consisting of biotin, streptavidin, and combinations thereof and the second binding molecule is selected from the group consisting of streptavidin, biotin, and combinations thereof. 
     
     
         48 . The method of  claim 41  wherein the molar ratio of the methylation-specific hybridization probes to the corresponding unmethylated competitor probes is from about 0.02:1 to about 10:1.