Methylation analysis using nicking agents

• US 6,884,586 B2
• Filed: 07/15/2002
• Issued: 04/26/2005
• Est. Priority Date: 07/15/2001
• Status: Active Grant

• Current Assignee: Keck Graduate Institute
• Original Assignee: Keck Graduate Institute
• Inventors: Van Ness, Lori K., Van Ness, Jeffrey, Galas, David J.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): CALAMITA, HEATHER
• Application Number:
  US10/197,616
• Publication Number:
  US 20030152952A1
• Time in Patent Office: 1,016 Days
• Field of Search: 435/5, 435/6, 435/91.2, 435/320.1, 435/69.1, 536/23.1, 536/23.4
• US Class Current: 435/6.14
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The present invention provides methods and compositions for nucleic acid methylation analysis using nicking agents.

Claims

1. A method for characterizing the methylation state of a target nucleic acid, comprising:

a. treating the target nucleic acid with a modifying agent that differentially modifies a nucleotide based on the methylation state of the nucleotide to provide a treated target nucleic acid;

b. providing a template double-stranded nucleic acid that comprises a nicking agent recognition sequence (NARS) and a portion of the treated target nucleic acid or an amplification product thereof if the target nucleic acid is single-stranded or a portion of one strand of the treated target nucleic acid if the target nucleic acid is double-stranded;

c. amplifying a single-stranded nucleic acid fragment in the presence of a nicking agent (NA) that recognizes the NARS, a DNA polymerase, and one or more deoxynucleoside triphosphate(s), wherein the amplifying uses a portion of the template double-stranded nucleic acid as a template, wherein the single-stranded nucleic acid fragment contains no more than 20 nucleotides; and

d. characterizing the single-stranded nucleic acid fragment and thereby characterizing the methylation state of the target nucleic acid.

2. The method of claim 1 wherein the modifying agent is a bisulfite salt.

3. The method of claim 2 wherein the modifying agent is sodium bisulfite.

4. The method of claim 1 wherein the NA is a nicking endonuclease (NE).

5. The method of claim 4 wherein the NE is N.BstNB I.

6. The method of claim 1 wherein the target nucleic acid is single-stranded.

7. The method of claim 6 wherein the target nucleic acid is one strand of a denatured double-stranded nucleic acid.

8. The method of claim 1 wherein the DNA polymerase is selected from the group consisting of exo⁻

Vent DNA polymerase, exo⁻

Deep Vent DNA polymerase, exo⁻

Bst DNA polymerase, 9^oN_m™

DNA polymerase and exo⁻

Bca DNA polymerase.

9. The method of claim 1 wherein the characterizing is performed at least partially by the technique selected from the group consisting of spectrometry, chromatography, and electrophoresis.

10. The method of claim 1 wherein the providing is performed by the steps, comprising

(1) forming a mixture comprising the treated target nucleic acid, a first oligonucleotide primer (ODNP) and a second ODNP, wherein

(a) if the target nucleic acid is single-stranded,

the first ODNP comprises a sequence of a sense strand of a NARS, and a nucleotide sequence at least substantially complementary to a first nucleotide sequence of the target nucleic acid,

the second ODNP comprises a sequence at least substantially identical to a second nucleotide sequence of the target nucleic acid, the second nucleotide sequence located 5′

to the first nucleotide sequence;

or

(b) if the target nucleic acid is double-stranded having a first strand and a second strand,

the first ODNP comprises a sequence of a sense strand of a NARS and a nucleotide sequence at least substantially complementary to a first nucleotide sequence of the first strand of the target nucleic acid,

the second ODNP comprises a sequence at least substantially complementary to a second nucleotide sequence of the second strand of the target nucleic acid, the nucleotide sequence in the first strand of the target nucleic acid that corresponds to the second nucleotide sequence in the second strand of the target nucleic acid being located at 5′

to the first nucleotide sequence in the first strand of the target nucleic acid; and

(2) maintaining said mixture at conditions that amplify a template double-stranded nucleic acid that comprises the NARS.

11. The method of claim 1 wherein the providing is performed by the steps, comprising

(a) if the target nucleic acid is single-stranded,

(i) synthesizing a completely complementary strand of the treated target nucleic acid to provide a double-stranded nucleic acid fragment, and

(ii) ligating an adaptor to the double-stranded nucleic acid fragment of step (i), wherein the adaptor comprises a NARS;

or

(b) if the target nucleic acid is double-stranded,

(i) ligating an adaptor to the treated target nucleic acid, wherein the adaptor comprises a NARS.

12. The method of claim 1 wherein the NA is a restriction endonuclease (RE).

13. The method of claim 1 wherein the amplifying is performed in the presence of a strand displacement facilitator.

14. The method of claim 13 wherein the strand displacement facilitator is trehalose.

15. The method of claim 9 wherein the spectrometry is mass spectrometry.

16. The method of claim 9 wherein the chromatography is liquid chromatography.

17. The method of claim 9 wherein the characterizing is performed at least partially by liquid chromatography and mass spectrometry.

18. The method of claim 10 wherein

(i) if the target nucleic acid is single-stranded, the first ODNP comprises a sequence completely complementary to the first nucleotide sequence of the target nucleic acid;

or

(ii) if the target nucleic acid is double-stranded, the first ODNP comprises a sequence completely complementary to the first nucleotide sequence of the first strand of the target nucleic acid.

19. The method of claim 10 wherein

(i) if the target nucleic acid is single-stranded, the second ODNP comprises a sequence completely identical to the second nucleotide sequence of the target nucleic acid;

or

(ii) if the target nucleic acid is double-stranded, the second ODNP comprises a sequence completely complementary to the second nucleotide sequence of the second strand of the target nucleic acid.

20. The method of claim 10 wherein the first ODNP or the second ODNP is immobilized.

21. The method of claim 11 wherein the adaptor further comprises a type IIs restriction endonuclease recognition sequence (TRERS), wherein the nicking site in the template nucleic acid of a NA that recognizes the NARS is located 5′

to the position corresponding to the cleavage site of a type IIs restriction endonuclease that recognizes the TRERS in the strand of the template nucleic acid that does not contain the nicking site.

22. The method of claim 21, wherein the type IIs restriction endonuclease is Bmp I.

23. The method of claim 1 wherein the single-stranded nucleic acid fragment contains no more than 17 nucleotides.

24. The method of claim 1 wherein the single-stranded nucleic acid fragment contains no more than 12 nucleotides.

25. The method of claim 11 wherein the adaptor is immobilized to a solid support.

26. The method of claim 1 wherein the providing is performed by the steps, comprising

(1) forming a mixture comprising

(A) the treated target nucleic acid, and

(B) an oligonucleotide primer that

i) comprises a sequence of the sense strand of a NARS, and

ii) is at least substantially complementary to a first portion of the treated target nucleic acid or to a first portion of one strand of the target nucleic acid;

and

(2) extending the oligonucleotide primer using

(A) a second portion of the treated single-stranded target nucleic acid located 5′

to the first portion of the single-stranded target nucleic acid, or

(B) a second portion of the one strand of the treated double-stranded nucleic acid located 5′

to the first portion of the one strand of the treated double-stranded nucleic acid

as a template to provide the template nucleic acid.

27. The method of claim 26 wherein one or more nucleotides in the sense strand of the NARS do not form a conventional base pair with nucleotides of the treated single-stranded target nucleic acid or of the one strand of the treated double-stranded target nucleic acid.

28. The method of claim 26 wherein all the nucleotides in the sense strand of the NARS form conventional base pairs with nucleotides of the treat single-stranded target nucleic acid or of the one strand of the treated double-stranded target nucleic acid.

29. The method of claim 1 wherein the providing is performed by the steps, comprising

(1) forming a mixture comprising

(A) the treated target nucleic acid,

(B) an oligonucleotide primer that

i) comprises a sequence of the sense strand of a NARS, and

ii) is at least substantially complementary to a first portion of the treated target nucleic acid or to a first portion of one strand of the target nucleic acid;

and

(C) a partially double-stranded nucleic acid that

i) comprises a double-stranded type IIs restriction endonuclease recognition sequence,

ii) a 3′

overhang that

a) is at least substantially complementary to a second region of the single-stranded target nucleic acid located 5′

to the first region of the single-stranded target nucleic acid, or

b) is at least substantially complementary to a second region of the one strand of the double-stranded target nucleic acid located 5′

to the second region of the one strand of the double-stranded target nucleic acid,

under conditions that allow for hybridization between the oligonucleotide primer and the first region of the single-stranded target nucleic acid or of the one strand of the double-stranded nucleic acid and between the 3′

overhang of the partially double-stranded nucleic acid and the second region of the single-stranded target nucleic acid or of the one strand of the double-stranded nucleic acid;

(2) digesting the single-stranded target nucleic acid or the one strand of the double-stranded target nucleic acid that have hybridized to the oligonucleotide primer and to the partially double-stranded nucleic acid in the second region; and

(3) extending from the 3′

terminus of the oligonucleotide primer using the region between the first and second regions of the treated single-stranded target nucleic acid or between the first and second regions of the one strand of the treated double-stranded target nucleic acid as a template to provide the template nucleic acid.

30. The method of claim 29 wherein one or more nucleotides in the sense strand of the NARS do not form a conventional base pair with nucleotides of the treated single-stranded target nucleic acid or of the one strand of the treated double-stranded target nucleic acid.

31. The method of claim 29 wherein all the nucleotides in the sense strand of the NARS form conventional base pairs with nucleotides of the treated single-stranded target nucleic acid or of the one strand of the treated double-stranded target nucleic acid.

32. The method of claim 1 wherein the target nucleic acid is immobilized to a solid support.

33. A method for the multiplex characterization of methylation states of at least portions of target nucleic acids, comprising

a. treating the target nucleic acids with a modifying agent that differentially modifies a nucleotide based on the methylation state of the nucleotide, to provide treated target nucleic acids;

b. for each target nucleic acid, providing a template double-stranded nucleic acid that comprises a nicking agent recognition sequence (NARS) and a portion of the treated target nucleic acid or an amplification product thereof;

c. amplifying single-stranded nucleic acid fragments in the presence of a nicking agent (NA) that recognizes the NARS, a DNA polymerase, and one or more deoxynucloside triphosphate(s), wherein the amplifying uses a portion of each template double-stranded nucleic acid as a template, wherein the single-stranded nucleic acid fragments each contains no more than 20 nucleotides; and

d. characterizing the single-stranded nucleic acid fragments and thereby characterizing the methylation state of at least portions of the target nucleic acids.

34. The method of claim 33 wherein the modifying agent is a bisulfite salt.

35. The method of claim 34 wherein the modifying agent is sodium bisulfite.

36. The method of claim 33 wherein the NA is a nicking endonuclease (NE).

37. The method of claim 36 wherein the NE is N.BstNB I.

38. The method of claim 33 wherein the single-stranded target nucleic acid is one strand of a denatured double-stranded nucleic acid.

39. The method of claim 33 wherein the DNA polymerase is selected from the group consisting of exo⁻

Vent DNA polymerase, exo⁻

Deep Vent DNA polymerase, exo⁻

Bst DNA polymerase, 9^oN_m™

DNA polymerase, and exo⁻

Bca DNA polymerase.

40. The method of claim 33 wherein the characterizing is performed at least partially by mass spectrometry.

41. The method of claim 33 wherein the charactering is performed at least partially by liquid chromatography and mass spectrometry.

42. The method of claim 33 wherein the single-stranded nucleic acid fragments each contains no more than 17 nucleotides.

43. The method of claim 33 wherein the single-stranded nucleic acid fragments each contains no more than 12 nucleotides.

44. A method for characterizing the methylation state of a target nucleic acid, comprising:

a. treating the target nucleic acid with a modifying agent that differentially modifies a nucleotide based on the methylation state of the nucleotide to provide a treated target nucleic acid;

b. providing a first template double-stranded nucleic acid that comprises a nicking agent recognition sequence (NARS) and a portion of the treated target nucleic acid or an amplification product thereof if the target nucleic acid is single-stranded or a portion of one strand of the treated target nucleic acid if the target nucleic acid is double-stranded;

c. providing a second template double-stranded nucleic acid that comprises the NARS and a portion of untreated target nucleic acid or an amplification product thereof if the target nucleic acid is single-stranded or a portion of one strand of the untreated target nucleic acid if the target nucleic acid is double-stranded;

d. amplifying a first single-stranded nucleic acid fragment in the presence of a nicking agent (NA) that recognizes the NARS, a DNA polymerase, and one or more deoxynucleoside triphosphate(s), wherein the amplifying uses a portion of the first template double-stranded nucleic acid as a template, wherein the first single-stranded nucleic acid fragment contains no more than 20 nucleotides;

e. amplifying a second single-stranded nucleic acid fragment in the presence of of the NA, the DNA polymerase and the deoxynucleoside triphosphate(s), wherein the amplifying uses a portion of the second template double-stranded nucleic acid as a template, wherein the second single-stranded nucleic acid fragment contains no more than 20 nucleotides;

f. characterizing the first single-stranded nucleic acid fragment;

g. characterizing the second single-stranded nucleic acid fragment; and

h. comparing the characteristic of the first single-straned nucleic acid fragment obtained from step (f) with the characteristic of the second single-stranded nucleic acid fragment obtained from step (g), and thereby characterizing the methylation state of the target nucleic acid.

45. The method of claim 44 wherein the modifying agent is a bisulfite salt.

46. The method of claim 45 wherein the modifying agent is sodium bisulfite.

47. The method of claim 44 wherein the NA is a nicking endonuclease (NE).

48. The method of claim 47 wherein the NE is N.BstNB I.

49. The method of claim 44 wherein step (f) and step (g) are performed at least partially by the technique selected from the group consisting of spectrometry, chromatography, and electrophoresis.