Process for high throughput DNA methylation analysis

• US 7,112,404 B2
• Filed: 12/10/2001
• Issued: 09/26/2006
• Est. Priority Date: 05/14/1999
• Status: Expired due to Term

• Current Assignee: University of Southern California
• Original Assignee: Epigenomics AG
• Inventors: Eads, Cindy A., Danenberg, Kathleen D., Laird, Peter W.
• Primary Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US10/016,505
• Publication Number:
  US 20020086324A1
• Time in Patent Office: 1,751 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/25.32, 536/24.33
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)

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.

Claims

1. 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, in real-time during the amplification, the methylated nucleic acid based on amplification-mediated probe displacement.

2. The method of claim 1 wherein the amplifying step is a polymerase chain reaction (PCR).

3. The method of claim 1 wherein the modifying agent is bisulfite.

4. The method of claim 1 wherein the converted nucleic acid contains uracil in place of unmethylated cytosine residues present in the unmodified nucleic acid-containing sample.

5. The method of claim 1 wherein the probe further comprises one or a plurality of fluorescence label moieties.

6. The method of claim 5 wherein the amplification and detection step comprises fluorescence-based quantitative PCR.

7. The method of claim 5, wherein the probe is a FRET probe, or a dual-label probe comprising a fluorescence-reporter moiety and fluorescence-quencher moiety.

8. The method of claim 7, wherein the FRET probe is one component of a real-time PCR hybridization probe pair.

9. The method of claim 7, wherein the probe is a nuclease cleavable dual-label probe.

10. The method of claim 1, wherein at least one of the primers comprises a CpG-speciflc probe.

11. The method of claim 1, wherein detecting based on amplification-mediated probe displacement, comprises detecting amplification-mediated change of probe fluorescence.

12. 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, in real-time during the amplification, the methylated nucleic acid based on amplification-mediated probe displacement.

13. The method of claim 12 wherein the amplifying step comprises a polymerase chain reaction (PCR).

14. The method of claim 12 wherein the modifying agent comprises bisulfite.

15. The method of claim 12 wherein the converted nucleic acid contains uracil in place of unmethylated cytosine residues present in the unmodified nucleic acid-containing sample.

16. The method of claim 12 wherein the probe further comprises one or a plurality of fluorescence label moieties.

17. The method of claim 16 wherein the amplification and detection step comprises fluorescence-based quantitative PCR.

18. The method of claim 16, wherein the probe is a FRET probe, or a dual-label probe comprising a fluorescence-reporter moiety and fluorescence-quencher moiety.

19. The method of claim 18, wherein the FRET probe is one component of a real-time PCR hybridization probe pair.

20. The method of claim 18, wherein the probe is a nuclease cleavable dual-label probe.

21. The method of claim 12, wherein at least one of the primers comprises a CpG-specific probe.

22. The method of claim 12, wherein detecting based on amplification-mediated probe displacement, comprises detecting amplification-mediated change of probe fluorescence.

23. The method of claim 12 wherein methylation amounts in the nucleic acid sample are quantitatively determined based on reference to a control reaction for amount of input nucleic acid.

24. 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, in real-time during the amplification, the methylated nucleic acid based on amplification-mediated probe displacement.

25. The method of claim 24 wherein the amplifying step comprises a polymerase chain reaction (PCR).

26. The method of claim 24 wherein the modifying agent is bisulfite.

27. The method of claim 24 wherein the converted nucleic acid contains uracil in place of unmethylated cytosine residues present in the unmodified nucleic acid-containing sample.

28. The method of claim 24 wherein the probe further comprises one or a plurality of fluorescence label moieties.

29. The method of claim 28 wherein the amplification and detection step comprises fluorescence-based quantitative PCR.

30. The method of claim 28, wherein the probe is a FRET probe, or a dual-label probe comprising a fluorescence-reporter moiety and fluorescence-quencher moiety.

31. The method of claim 30, wherein the FRET probe is one component of a real-time PCR hybridization probe pair.

32. The method of claim 30, wherein the probe is a nuclease cleavable dual-label probe.

33. The method of claim 24, wherein at least one of the primers comprises a CpG-specific probe.

34. The method of claim 24, wherein detecting based on amplification-mediated probe displacement, comprises detecting amplification-mediated change of probe fluorescence.

35. A method for detecting cytosine methylation within a genomic sample of DNA, comprising:

obtaining a methylation kit;

conducting, using the kit, a real-time methylation assay of the genomic DNA sample; and

determining, based on the methylation assay, whether cytosine methylation is present in the DNA sample, and wherein the methylation kit comprises;

a modifying agent that modifies unmethylated cytosine to produce a converted nucleic acid;

primers for amplification of the converted nucleic acid;

primers for the amplification of control unmodified nucleic acid; and

a CpG-specific probe the detection of which, during the amplification of the converted nucleic acid, is in real-time based on amplification-mediated probe displacement, wherein the CpG-specific probe distinguishes between modified unmethylated and methylated nucleic acid, and wherein the primers each may or may not distinguish between unmethylated and methylated nucleic acid.

36. The method of claim 35, wherein the modifying agent is bisulfite.

37. The method of claim 35 wherein the modifying agent converts cytosine residues to uracil residues.

38. The method of claim 35, wherein the CpG-speciflc probe, but not the primers for amplification of the converted nucleic acid, distinguishes between modified unmethylated and methylated nucleic acid.

39. The method of claim 35, wherein both the CpG-specific probe, and the primers for amplification of the converted nucleic acid, distinguish between modified unmethylated and methylated nucleic acid.

40. The method of claim 35, wherein the CpG-specific probe further comprises one or a plurality of fluorescence label moieties.

41. The method of claim 35, wherein one of the primers for amplification of the converted nucleic acid comprises the CpG-specific probe.

42. The method of claim 40, wherein the CpG-specific probe is a FRET probe, a real-time PCR hybridization probe, or a dual-label probe.

43. The method of claim 35, wherein detecting based on amplification-mediated probe displacement, comprises detecting amplification-mediated change of probe fluorescence.