Methylation analysis using nicking agents

• US 20030152952A1
• Filed: 07/15/2002
• Published: 08/14/2003
• Est. Priority Date: 07/15/2001
• Status: Active Grant

• Current Assignee: Keck Graduate Institute
• Original Assignee: Keck Graduate Institute
• Inventors: Van Ness, Jeffrey, Galas, David J., Van Ness, Lori K.
• Granted Patent: US 6,884,586 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 necleotide 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; and

d. characterizing the single-stranded nucleic acid fragment and thereby characterizing the methylation state of the single-stranded 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 NA is a restriction endonuclease (RE).

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

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

9. The method of claim 1 wherein the single-stranded target nucleic acid is derived from a eukaryotic organism.

10. The method of claim 9 wherein the single-stranded target nucleic acid is derived from a human.

11. 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.

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

13. The method of claim 12 wherein the strand displacement facilitator is trehalose.

14. 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.

15. The method of claim 14 wherein the spectrometry is mass spectrometry.

16. The method of claim 14 wherein the chromatography is liquid chromatography.

17. The method of claim 14 wherein the characterizing is performed at least partially by liquid chromatography and mass spectrometry.

18. 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.

19. The method of claim 18 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.

20. The method of claim 18 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.

21. The method of claim 18 wherein the first ODNP, the second ODNP, or both ODNPs are immobilized.

22. 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.

23. The method of claim 22 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.

24. The method of claim 23 wherein the type IIs restriction endonuclease is Bmp I.

25. The method of claim 1 wherein the single-stranded nucleic acid fragment contains no more than 20 nucleotides.

26. The method of claim 25 wherein the single-stranded nucleic acid fragment contains no more than 17 nucleotides.

27. The method of claim 26 wherein the single-stranded nucleic acid fragment contains no more than 12 nucleotides.

28. The method of claim 22 wherein the adaptor is immobilized to a solid support.

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, 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.

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 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.

32. The method of claim 31 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.

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

34. 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; and

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

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

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

37. The method of claim 34 wherein the NA is a nicking endonuclease (NE).

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

39. The method of claim 34 wherein the single-stranded target nucleic acid is one strand of a denatured double-stranded nucleic acid.

40. The method of claim 34 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.

41. The method of claim 34 wherein the characterizing is performed at least partially by mass spectrometry.

42. The method of claim 34 wherein the charactering is performed at least partially by liquid chromatography and mass spectrometry.

43. The method of claim 34 wherein the single-stranded nucleic acid fragments each contains no more than 20 nucleotides.

44. The method of claim 43 wherein the single-stranded nucleic acid fragments each contains no more than 17 nucleotides.

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

46. An isolated nucleic acid comprising:

(1) a nicking agent recognition sequence (NARS); and

(2) a double-stranded nucleic acid fragment that comprises;

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

(i) the target nucleic acid that has been treated and modified by a modifying agent that differentially modifies a nucleotide based on the methylation state of the nucleotide, and

(ii) a nucleotide sequence that is completely complementary to the target nucleic acid that has been treated and modified by the modifying agent;

wherein in the strand of said nucleic acid that contains the sense strand of the NARS, the sequence of the sense strand of the NARS is located 5′

to nucleotide sequence (ii);

or

(b) if a target nucleic acid is double-stranded, the target nucleic acid that either said target nucleic acid or one strand thereof has been treated and modified by a modifying agent that differentially modifies a nucleotide based on the methylation state of the nucleotide,

wherein in the strand of said nucleic acid that contains the sense strand of the NARS, the sequence of the sense strand of the NARS is located 5′

to the nucleotide sequence of one strand of the target nucleic acid that has been treated and modified by the modifying agent.

47. The nucleic acid of claim 46 wherein the modifying agent is a bisulfite salt.

48. The nucleic acid of claim 47 wherein the modifying agent is sodium bisulfite.

49. The nucleic acid of claim 46 wherein the NARS is recognizable by a nicking endonuclease (NE).

50. The nucleic acid of claim 49 wherein the nicking endonuclease is N.BstNB I.

51. The nucleic acid of claim 46 wherein a single-stranded nucleic acid fragment amplified using the double-stranded nucleic acid as a template in the presence of a NA that recognizes the NARS contains no more than 20 nucleotides.

52. The nucleic acid of claim 51 wherein the single-stranded nucleic acid fragment contains no more than 17 nucleotides.

53. The nucleic acid of claim 51 wherein the single-stranded nucleic acid fragments contains no more than 12 nucleotides.

54. The nucleic acid of claim 46 wherein the nucleic acid is immobilized.

55. A composition comprising:

a. a single-stranded target nucleic acid that has been treated and modified by a modifying agent that modifies a nucleotide based on the methylation state of the nucleotide;

b. a first oligonucleotide primer (ODNP) that comprises a sequence of a sense strand of a nicking agent recognition sequence (NARS) and a sequence that is at least substantially complementary to a first nucleotide sequence of the target nucleic acid; and

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

to the first nucleotide sequence.

56. The composition of claim 55 wherein the first ODNP comprises a sequence that is completely complementary to the first nucleotide sequence of the target nucleic acid.

57. The composition of claim 55 wherein the second ODNP comprises a sequence that is completely identical to the second nucleotide sequence of the target nucleic acid.

61. The composition of claim 55 or 58 further comprising a nicking agent (NA) that recognizes the NARS.

62. The composition of claim 55 or 58 wherein the NA is a nicking endonuclease (NE).

63. The composition of claim 62 wherein the NE is N.BstNB I.

64. The composition of claim 61 wherein the NA is a restriction endonuclease (RE).

65. The composition of claim 61 further comprising a DNA polymerase.

66. The composition of claim 65 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.

67. The composition of claim 66 further comprising a stand displacement facilitator.

68. The composition of claim 67 wherein the strand displacement facilitator is trehalose.

69. The composition of claim 67 further comprising one or more deoxynucleoside triphosphate(s).

70. The composition of claim 69 wherein a single-stranded nucleic acid fragment amplified using at least a portion of the target nucleic acid that has been treated with the modifying agent or an amplification product thereof as a template contains no more than 20 nucleotides.

71. The composition of claim 70 wherein the single-stranded nucleic acid fragment contains no more than 17 nucleotides.

72. The composition of claim 70 wherein the single-stranded nucleic acid fragments contains no more than 12 nucleotides.

58. A composition comprising:

a. a double-stranded target nucleic acid that has been treated and modified by a modifying agent that modifies a nucleotide based on the methylation state of the nucleotide, the double-stranded target nucleic acid having a first strand and a second strand;

b. a first oligonucleotide primer (ODNP) that comprises a sequence of a sense strand of a nicking agent recogntion sequence (NARS) and a sequence that is at least substantially complementary to a first nucleotide sequence of the first strand of the target nucleic acid; and

c. a second ODNP that comprises a sequence that is at least substantially complementary to a second nucleotide sequence of the second strand of the target nucleic acid, wherein the nucleotide sequence in the first strand of the target nucleic acid that corresponding to the second nucleotide sequence in the second strand of the target nucleic acid is located at 5′

to the first nucleotide sequence in the first strand of the target nucleic acid.

59. The composition of claim 58 wherein the first ODNP comprises a sequence that is completely complementary to the first nucleotide sequence of the first strand of the target nucleic acid.

60. The composition of claim 58 wherein the second ODNP comprise a sequence that is completely complementary to the second nucleotide sequence of the second strand of the target nucleic acid.