Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
AssigneeThe Johns Hopkins University School of Medicine
InventorsJames G. Herman, Stephen B. Baylin
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Abstract
The present invention provides a method of PCR, methylation specific PCR (MSP), for rapid identification of DNA methylation patterns in a CpG-containing nucleic acid. MSP uses agents to modify unmethylated cytosine in a nucleic acid of interest, and then uses the PCR reaction to amplify the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers. The oligonecleotide primers distinguish between modified methylated and nonmethylated nucleic acid. Kits utilizing MSP for the detection of methylated CpG-containing nucleic acids are also provided.
Claims27
A method for detecting a methylated CpG-containing nucleic acid comprising:
- contacting a nucleic acid-containing specimen with an agent that modifies unmethylated cytosine,
- amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and
- detecting the methylated nucleic acid based on the presence or absence of amplification products produced in said amplifying step.
A kit useful for the detection of a methylated CpG-containing nucleic acid comprising carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of the CpG-containing nucleic acid, wherein the primers distinguish between modified methylated and nonmethylated nucleic acid.
Isolated oligonucleotide primer(s) for detection of a methylated CpG-containing nucleic acid wherein the primer hybridizes with a target polynucleotide sequence having the sequence selected from the group consisting of SEQ ID NO:1-23 and SEQ ID NO:24.
A kit for the detection of methylated CpG-containing nucleic acid from a sample comprising:
- a) a reagent that modifies unmethylated cytosine nucleotides;
- b) a wild-type unmodified control nucleic acid;
- c) primers for the amplification of unmethylated CpG-containing nucleic acid;
- d) primers for the amplification of methylated CpG-containing nucleic acid;
- and
- e) primers for the amplification of control unmodified nucleic acid,
- wherein the primers for the amplification of unmethylated CpG-containing nucleic acid and methylated CpG-containing nucleic acid distinguish between modified methylated and nonmethylated nucleic acid.
A method for detecting a methylated CpG-containing nucleic acid comprising:
- contacting a nucleic acid-containing specimen with bisulfite to modify unmethylated cytosine,
- amplifying the CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid and
- detecting the methylated nucleic acid based on the presence or absence of amplification products produced in said amplifying step.
Dependencies are inferred from the claim text.