Nucleic acid detection assay

• US 20070178459A1
• Filed: 09/03/2004
• Published: 08/02/2007
• Est. Priority Date: 09/04/2003
• Status: Active Grant

• Current Assignee: Human Genetic Signatures Pty., Ltd.
• Original Assignee: Human Genetic Signatures Pty., Ltd.
• Inventors: Millar, Douglas, Melki, John, Miklos, George
• Granted Patent: US 7,846,693 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

A method for determining the methylation status of a potential methylation site in genomic nucleic acid comprising treating genomic nucleic acid with an agent which modifies cytosine bases but does not modify 5methyl-cytosine bases under conditions to form a. modified nucleic acid template containing a potential methylation site; providing a first clamp containing a first capture sequence complementary to a region flanking one side of the potential methylation site in the modified nucleic acid template, providing a second clamp containing a second capture sequence complementary to a region flanking the other side of the potential methylation site in the modified nucleic acid template; allowing the first clamp and the second clamp to hybridise to the modified nucleic acid template; ligating the hybridised first and second clamps to form a probe spanning the potential methylation site in the modified nucleic acid template; digesting the modified nucleic acid template to obtain the probe; and detecting the probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.

Claims

1. A method for determining the methylation status of a potential methylation site in genomic nucleic acid comprising:

treating genomic nucleic acid with an agent which modifies cytosine bases but does not modify 5-methyl-cytosine bases under conditions to form a modified nucleic acid template from two complementary strands of genomic nucleic acid containing a potential methylation site;

providing a first clamp containing a first capture sequence and a second capture sequence, first capture sequence being complementary to a region flanking one side of the potential methylation site in the first strand of the modified nucleic acid template, and the second capture sequence being complementary to a region flanking one side of the potential methylation site in the second strand of the modified nucleic acid template;

providing a second clamp containing a third capture sequence complementary to a region flanking the other side of the potential methylation site in the modified nucleic acid template;

allowing the first clamp and the second clamp to hybridise to the modified nucleic acid template;

ligating the hybridised first and second clamps to form a probe spanning the potential methylation site in the modified nucleic acid template;

digesting the modified nucleic acid template to obtain the probe; and

detecting the probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.

2. The method according to claim 1 wherein the first clamp and the second clamp hybridise to the modified nucleic acid template such that there is no complementary bases at the potential methylation site and causing at least one base to be inserted between the first clamp and the second clamp prior to ligating the clamps to form a probe spanning the potential methylation site in the modified nucleic acid template.

3. The method according to claim 1 wherein the potential methylation site is cytosine (C) flanked 3′

by a guanine (G).

4. The method according to claim 1 wherein the modifying agent is selected from the group consisting of bisulfite, acetate, and citrate.

5. The method according to claim 4 wherein the modifying agent is sodium bisulfite.

6. The method according to claim 1 wherein at least one of the first or second clamps contains a universal primer which allows amplification of the probe after the ligation step.

7. The method according to claim 1 wherein at least one of the first or second clamps contains a capture site which allows capture of the probe.

8. The method according to claim 1 wherein at least one of the first or second clamps contains a cleavable site.

9. The method according to claim 8 wherein the cleavable site is nuclease restriction site.

10. The method according to claim 9 wherein the cleavable site is a uracil base.

11. The method according to claim 1 wherein the complementary sequences of the first or second clamps when ligated span the potential methylation site on the modified nucleic acid template.

12. The method according claim 1 wherein the clamps are ligated using a suitable enzyme capable of ligating single stranded nucleic acid.

13. The method according to claim 1 wherein the clamps are ligated at or near the first and second capture sequences.

14. The method according claim 2 wherein one or more bases are inserted between the clamps using a nucleic acid polymerase.

15. The method according to claim 14 wherein the base insertion reaction is carried out in the presence of a single nucleotide type and each nucleotide type is reacted separately such that the insertion of a particular base will be indicative of the identity of the complementary base on the modified nucleic acid template.

16. The method according to claim 1 wherein the modified nucleic acid template is digested by an enzyme.

17. The method according to claim 16 wherein the enzyme is Uracil N DNA Glycosylase.

18. The method according to claim 1 wherein the probe is detected and the methylation status of the potential methylation site in the modified genomic nucleic acid determined with a detection system which can recognise incorporation of the missing base(s) between the first and second clamps, or detect specific amplification of the probe.

19. The method according to claim 18 wherein the detection system is selected from the group consisting of hybridization by array, Southern blot type detection, Real-Time PCR quantitation, fluorescent beads, enzyme conjugates, radioactive beads, ligase chain reaction, and isothermal DNA amplification.

20. The method according to claim 1 wherein the genomic nucleic acid is DNA.

41. The method according to claim 19, wherein the isothermal DNA amplification is strand displacement amplification or rolling circle amplification.

21. A method for determining the methylation status of a potential methylation site on genomic nucleic acid comprising:

treating genomic nucleic acid with an agent which modifies cytosine bases but does not modify 5-methyl-cytosine bases under conditions to form a modified nucleic acid template from two complementary strands of genomic nucleic acid containing a potential methylation site;

providing a first clamp containing a first capture sequence and a second capture sequence, first capture sequence being complementary to a region flanking one side of the potential methylation site in the first strand of the modified nucleic acid template, and the second capture sequence being complementary to a region flanking one side of the potential methylation site in the second strand of the modified nucleic acid template;

providing a second clamp containing a third capture sequence and a fourth capture sequence, the third capture sequence being complementary to a region flanking the other side the potential methylation site in the first strand of the modified nucleic acid template, and the fourth capture sequence being complementary to a region flanking the other side of the potential methylation site in the second strand of the modified nucleic acid template;

allowing the first clamp and the second clamp to hybridise to the two complementary strands of the modified nucleic acid template;

ligating the hybridised clamps to form a circular probe spanning the potential methylation site in the complementary strands of the modified nucleic acid template;

digesting the modified nucleic acid template to obtain the circular probe; and

detecting the circular probe and determining the methylation status of the potential methylation site in the modified genomic nucleic acid.

22. The method according to claim 21 wherein the first clamp and the second clamp hybridise to the two complementary strands of the modified nucleic acid template such that there is no complementary bases at the potential methylation site of at least one of the complementary strands of the modified nucleic acid template, and causing at least one base to be inserted between the first clamp and the second clamp prior to ligating the clamps to form a probe spanning the potential methylation site in the modified nucleic acid template.

23. The method according to claim 21 wherein the potential methylation site is cytosine (C) flanked 3′

by a guanine (G).

24. The method according to claim 21 wherein the modifying agent is selected from the group consisting of bisulfite, acetate, and citrate.

25. The method according to claim 23 wherein the modifying agent is sodium bisulfite.

26. The method according to claim 21 wherein at least one of the first or second clamps contains a universal primer which allows amplification of the probe after the ligation step.

27. The method according to claim 21 wherein at least one of the first or second clamps contains a capture site which allows capture of the probe.

28. The method according to claim 21 wherein at least one of the first or second clamps contains a cleavable site.

29. The method according to claim 28 wherein the cleavable site is nuclease restriction site.

30. The method according to claim 28 wherein the cleavable site is a uracil base.

31. The method according to claim 21 wherein the complementary sequences of the first or second clamps when ligated span the potential methylation site on the modified nucleic acid template.

32. The method according to claim 21 wherein the clamps are ligated using a suitable enzyme capable of ligating single stranded nucleic acid.

33. The method according to claim 21 wherein the clamps are ligated at or near the first and second capture sequences.

34. The method according to claim 22 wherein one or more bases are inserted between the clamps using a DNA polymerase.

35. The method according to claim 34 wherein the base insertion reaction is carried out in the presence of a single nucleotide type and each nucleotide type is reacted separately such that the insertion of a particular base will be indicative of the identity of the complementary base on the modified nucleic acid template.

36. The method according to claim 21 wherein the modified nucleic acid template is digested by an enzyme.

37. The method according to claim 36 wherein the enzyme is Uracil N DNA Glycosylase.

38. The method according to claim 21 wherein the probe is detected and the methylation status of the potential methylation site in the modified genomic nucleic acid determined with a detection system which can recognise incorporation of the missing base(s) between the first and second clamps, or detect specific amplification of the probe.

39. The method according to claim 38 wherein the detection system is selected from the group consisting of hybridization by array, Southern blot type detection, Real-Time PCR quantitation, fluorescent beads, enzyme conjugates, radioactive beads, ligase chain reaction, and isothermal DNA amplification technologies including strand displacement amplification or rolling circle amplification.

40. The method according to claim 21 wherein the genomic nucleic acid is DNA.