Process for high-throughput DNA methylation analysis
AssigneeUniversity of Southern California
InventorsPeter W. Laird, Cindy A. Eads, Kathleen D. Danenberg
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Abstract
There is disclosed an improved high-throughput and quantitative process for determining methylation patterns in genomic DNA samples based on amplifying modified nucleic acid, and detecting methylated nucleic acid based on amplification-dependent displacement of specifically annealed hybridization probes. Specifically, the inventive process provides for treating genomic DNA samples with sodium bisulfite to create methylation-dependent sequence differences, followed by detection with fluorescence-based quantitative PCR techniques. The process is particularly well suited for the rapid analysis of a large number of nucleic acid samples, such as those from collections of tumor tissues.
Claims26
A method for detecting cytosine methylation and methylated CpG islands within a genomic sample of DNA comprising:
- (a) contacting a genomic sample of DNA with a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid;
- (b) amplifying the converted nucleic acid by means of oligonucleotide primers in the presence of one or a plurality of specific oligonucleotide probes, wherein one or a plurality of the oligonucleotide primers or the specific probe(s) are capable of distinguishing between unmethylated and methylated nucleic acid, with the proviso that at least one oligonucleotide probe is a CpG-specific probe capable of distinguishing between unmethylated and methylated nucleic acid;
- and
- (c) detecting the methylated nucleic acid based on amplification-mediated displacement of the CpG-specific probe.
A method for detecting a methylated CpG-containing nucleic acid comprising:
- (a) contacting a nucleic acid-containing sample with a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid;
- (b) amplifying the converted nucleic acid in the sample by means of oligonucleotide primers in the presence of a CpG-specific oligonucleotide probe, wherein the CpG-specific probe, but not the primers, distinguishes between modified unmethylated and methylated nucleic acid;
- and
- (c) detecting the methylated nucleic acid based upon an amplification-mediated displacement of the CpG-specific probe.
A method for detecting a methylated CpG-containing nucleic acid comprising:
- (a) contacting a nucleic acid-containing sample with a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid;
- (b) amplifying the converted nucleic acid in the sample by means of oligonucleotide primers in the presence of a CpG-specific oligonucleotide probe, wherein both the primers and the CpG-specific probe distinguish between modified unmethylated and methylated nucleic acid;
- and
- (c) detecting the methylated nucleic acid based on amplification-mediated displacement of the CpG-specific probe.
A methylation detection kit useful for the detection of a methylated CpG-containing nucleic acid comprising a carrier means being compartmentalized to receive in close confinement therein one or more containers comprising:
- (i) a first container containing a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid;
- (ii) a second container containing primers for amplification of the converted nucleic acid;
- (iii) a third container containing primers for the amplification of control unmodified nucleic acid;
- and
- (iv) a fourth container containing a specific oligonucleotide probe the detection of which is based on amplification-mediated displacement, wherein the specific oligonucleotide probe is a CpG-specific oligonucleotide probe that distinguishes between modified unmethylated and methylated nucleic acid, and wherein the primers may or may not distinguish between unmethylated and methylated nucleic acid.
Dependencies are inferred from the claim text.