5-methylcytosine detection, compositions and methods therefor

• US 20060172309A1
• Filed: 06/30/2004
• Published: 08/03/2006
• Est. Priority Date: 06/30/2004
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Zon, Gerald
• Granted Patent: US 7,399,614 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

Compositions and methods for detecting 5-methylcytosine in a nucleic acid are disclosed. A 5-methylcytosine discriminator, which is a deoxyribonucleosidetriphosphate comprising a cytosine-pairing moiety such as a guanosine and a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is described. The discriminator is able to base pair with a cytosine but not a 5-methylcytosine. A 5-methylcytosine comprised by a target nucleotide can be detected in a reaction using a DNA polymerase and a primer hybridized immediately adjacent to the target nucleotide. In the reaction, pyrophosphate released upon incorporation of a dNTP complementary to a target nucleotide is detected. Lack of incorporation of the discriminator, but incorporation of a dGTP, can indicate that the target nucleotide is a 5-methylcytosine.

Claims

1. A 5-methylcytosine discriminator comprising:

a cytosine-pairing moiety; and

a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.

2. A 5-methylcytosine discriminator according to claim 1, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is an alkyl moiety.

3. A 5-methylcytosine discriminator according to claim 1, wherein the alkyl moiety is a methyl moiety.

4. A 5-methylcytosine discriminator according to claim 1, further comprising a linker extending between the cytosine-pairing moiety and the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.

5. A 5-methylcytosine discriminator according to claim 4, wherein the linker is a planar linker.

6. A 5-methylcytosine discriminator according to claim 5, wherein the planar linker comprises a plurality of fused rings.

7. A 5-methylcytosine discriminator according to claim 6, wherein the plurality of fused rings comprises one or more aromatic rings.

8. A 5-methylcytosine discriminator according to claim 6, wherein the plurality of fused rings comprises from three to about ten fused rings.

9. A 5-methylcytosine discriminator according to claim 8, wherein the plurality of fused rings comprises from four to about eight fused rings.

10. A 5-methylcytosine discriminator according to claim 8, wherein the plurality of fused rings comprises three fused rings.

11. A 5-methylcytosine discriminator according to claim 9, wherein the plurality of fused rings comprises four fused rings.

12. A 5-methylcytosine discriminator according to claim 5, wherein the cytosine-pairing moiety and the planar linker are rigidly attached.

13. A 5-methylcytosine discriminator according to claim 5, wherein the cytosine-pairing moiety and the planar linker are co-planar.

14. A 5-methylcytosine discriminator according to claim 12, further comprising an amide bond extending between the cytosine-pairing moiety and the linker.

15. A 5-methylcytosine discriminator according to claim 1, comprising a structure

16. A 5-methylcytosine discriminator according to claim 1, comprising a structure

17. A 5-methylcytosine discriminator according to claim 1, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.6 Angstroms.

18. A 5-methylcytosine discriminator according to claim 1, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.2 Angstroms.

19. A 5-methylcytosine discriminator according to claim 1, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 0.75 Angstroms.

20. A 5-methylcytosine discriminator according to claim 1, wherein the cytosine-pairing moiety is a guanine moiety.

21. A 5-methylcytosine discriminator according to claim 1, further comprising a pentose moiety.

22. A 5-methylcytosine discriminator according to claim 21, wherein the pentose moiety is a 2′

-deoxyribose moiety.

23. A 5-methylcytosine discriminator according to claim 21, wherein the pentose moiety is a 2′

,3′

-dideoxyribose moiety.

24. A 5-methylcytosine discriminator according to claim 21, further comprising three phosphate moieties.

25. A 5-methylcytosine discriminator according to claim 1, comprising the structure:

26. A 5-methylcytosine discriminator according to claim 1, comprising the structure:

27. A 5-methylcytosine discriminator according to claim 1, further comprising a label.

28. A 5-methylcytosine discriminator according to claim 27, wherein the label is a fluorophore selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

29. A 5-methylcytosine discriminator according to claim 28, wherein the fluorophore is VIC or FAM.

30. A 5-methylcytosine discriminator according to claim 27, wherein the label is a hapten.

31. A 5-methylcytosine discriminator according to claim 30, wherein the hapten is biotin or digoxygenin.

32. A 5-methylcytosine discriminator according to claim 27, wherein the label is a radioisotope selected from ³²P, ³³P, ³⁵S, ³H and ¹⁴C.

33. A method of detecting 5-methylcytosine comprised by a target nucleic acid, the method comprising:

a) forming a detection mixture comprising a DNA polymerase and a hybrid nucleic acid comprising the target nucleic acid hybridized to a primer oligonucleotide, the primer oligonucleotide comprising a 3′

end nucleotide base paired with its complement in the target nucleic acid immediately adjacent to a target nucleotide;

b) adding a 5-methylcytosine discriminator to the detection mixture; and

c) detecting inhibition of hydrogen bond formation between the 5-methylcytosine discriminator and the target nucleotide.

34. A method of detecting 5-methylcytosine according to claim 33, wherein detecting inhibition of hydrogen bond formation between the 5-methylcytosine discriminator and target nucleotide comprises detecting inhibition of pyrophosphate release from the 5-methylcytosine discriminator.

35. A method of detecting 5-methylcytosine according to claim 34, wherein detecting inhibition of hydrogen bond formation between the 5-methylcytosine discriminator and target nucleotide further comprises adding dGTP to the detection mixture subsequent to the adding of 5-methylcytosine discriminator, and detecting pyrophosphate release from the dGTP.

36. A method of detecting 5-methylcytosine according to claim 33, wherein the primer oligonucleotide comprises at least about 10 nucleotides up to about 400 nucleotides.

37. A method of detecting 5-methylcytosine according to claim 33, wherein the primer oligonucleotide comprises at least about 15 nucleotides up to about 100 nucleotides.

38. A method of detecting 5-methylcytosine according to claim 33, wherein the primer oligonucleotide comprises at least about 20 nucleotides up to about 50 nucleotides.

39. A method of detecting 5-methylcytosine according to claim 33, wherein the primer oligonucleotide is at least about 70% complementary to a contiguous sequence of the target nucleic acid.

40. A method of detecting 5-methylcytosine according to claim 33, wherein the detection mixture further comprises a sulfurylase, a luciferase, and an apyrase.

41. A method of detecting 5-methylcytosine according to claim 33, wherein the 5-methylcytosine discriminator comprises a cytosine-pairing moiety and a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.

42. A method of detecting 5-methylcytosine according to claim 41, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is an alkyl moiety.

43. A method of detecting 5-methylcytosine according to claim 42, wherein the alkyl moiety is a methyl moiety.

44. A method of detecting 5-methylcytosine according to claim 41, wherein the 5-methylcytosine discriminator further comprises a planar linker extending between the cytosine-pairing moiety and the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.

45. A method of detecting 5-methylcytosine according to claim 44, wherein the planar linker comprises a plurality of fused rings.

46. A method of detecting 5-methylcytosine according to claim 44, wherein the 5-methylcytosine discriminator further comprises an amide bond extending between the cytosine-pairing moiety and the planar linker.

47. A method of detecting 5-methylcytosine according to claim 44, wherein the 5-methylcytosine discriminator comprises the structure

48. A method of detecting 5-methylcytosine according to claim 44, wherein the 5-methylcytosine discriminator comprises the structure

49. A method of detecting 5-methylcytosine according to claim 41, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.6 Angstroms.

50. A method of detecting 5-methylcytosine according to claim 41, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.2 Angstroms.

51. A method of detecting 5-methylcytosine according to claim 41, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 0.75 Angstroms.

52. A method of detecting 5-methylcytosine according to claim 41, wherein the cytosine-pairing moiety is a guanine moiety.

53. A method of detecting 5-methylcytosine according to claim 44, wherein the 5-methylcytosine discriminator comprises the structure:

54. A method of detecting 5-methylcytosine according to claim 44, wherein the 5-methylcytosine discriminator comprises the structure:

55. A method of detecting 5-methylcytosine according to claim 33, further comprising adding a dGTP or a ddGTP to the detection mixture, and wherein detecting inhibition of hydrogen bond formation between the 5-methylcytosine discriminator and the target nucleotide comprises detecting an elevated incorporation to the primer oligonucleotide of the dGTP or the ddGTP compared to incorporation to the primer oligonucleotide of the 5-methylcytosine discriminator.

56. A method of detecting 5-methylcytosine according to claim 55, wherein the 5-methylcytosine discriminator comprises a first label, and the dGTP or the ddGTP further comprises a second label different from the first label.

57. A method of detecting 5-methylcytosine according to claim 56, wherein the first label and the second label are each selected from the group consisting of a fluorophore, a hapten and a radioisotope.

58. A method of detecting 5-methylcytosine according to claim 57, wherein the fluorophore is selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

59. A method of detecting 5-methylcytosine according to claim 58, wherein the fluorophore is VIC or FAM.

60. A method of detecting 5-methylcytosine according to claim 56, wherein at least one of the first label and the second label is a hapten selected from biotin and digoxygenin.

61. A method of detecting 5-methylcytosine according to claim 56, wherein at least one of the first label and the second label is a radioisotope selected from ³²P, ³³P, ³⁵S, ³H and ¹⁴C.

62. A kit for detecting 5-methyl cytosine, the kit comprising:

a) a 5-methylcytosine discriminator, wherein the discriminator comprises a cytosine-pairing moiety and a moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine; and

b) packaging.

63. A kit for detecting 5-methyl cytosine according to claim 62, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine is an alkyl moiety.

64. A kit for detecting 5-methyl cytosine according to claim 62, wherein the alkyl moiety is a methyl moiety.

65. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator further comprises a planar linker comprising a plurality of fused aromatic rings extending between the cytosine-pairing moiety and the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine.

66. A kit for detecting 5-methyl cytosine according to claim 65, wherein the plurality of fused rings comprises three fused rings.

67. A kit for detecting 5-methyl cytosine according to claim 65, wherein the plurality of fused rings comprises four fused rings.

68. A kit for detecting 5-methyl cytosine according to claim 65, wherein the cytosine-pairing moiety and the planar linker are co-planar and wherein the 5-methylcytosine discriminator further comprises an amide bond extending between the cytosine-pairing moiety and the linker.

69. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator further comprises a structure

70. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator further comprises a structure

71. A kit for detecting 5-methyl cytosine according to claim 62, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.6 Angstroms.

72. A kit for detecting 5-methyl cytosine according to claim 62, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 1.2 Angstroms.

73. A kit for detecting 5-methyl cytosine according to claim 62, wherein the moiety which hinders hydrogen bonding between the cytosine-pairing moiety and a 5-methylcytosine, and a 5-methyl group of the 5-methylcytosine, have a calculated distance less than or equal to about 0.75 Angstroms.

74. A kit for detecting 5-methyl cytosine according to claim 62, wherein the cytosine-pairing moiety is a guanine moiety.

75. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator comprises the structure:

76. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator comprises the structure:

77. A kit for detecting 5-methyl cytosine according to claim 62, wherein the 5-methylcytosine discriminator further comprises a label.

78. A kit for detecting 5-methyl cytosine according to claim 77, wherein the label is a fluorophore selected from the group consisting of FAM, VIC, Sybra Green, TET, HEX, JOE, NED, LIZ, TAMRA, ROX, ALEXA, Texas Red, Cy3, Cy5, Cy7, Cy9, and dR6G.

79. A kit for detecting 5-methyl cytosine according to claim 78, wherein the fluorophore is FAM or VIC.

80. A kit for detecting 5-methyl cytosine according to claim 77, wherein the label is a radioisotope selected from ³²P, ³³P, ³⁵S ³H and ¹⁴C.

81. A kit for detecting 5-methyl cytosine according to claim 77, wherein the label is a hapten selected from biotin and digoxygenin.

82. A kit for detecting 5-methyl cytosine according to claim 77, further comprising a dGTP comprising a second label or a ddGTP comprising a second label.