Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
First Claim
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1. 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, anddetecting the methylated nucleic acid based on the presence or absence of amplification products produced in said amplifying step.
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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.
469 Citations
27 Claims
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1. A method for detecting a methylated CpG-containing nucleic acid comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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- 15. 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.
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20. 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. - View Dependent Claims (21)
- 1-23 and SEQ ID NO;
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22. A kit for the detection of methylated CpG-containing nucleic acid from a sample comprising:
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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. - View Dependent Claims (23, 24, 25, 26)
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27. A method for detecting a methylated CpG-containing nucleic acid comprising:
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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.
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Specification