Nucleotides and nucleosides and methods for their use in DNA sequencing

• US 8,148,503 B2
• Filed: 06/11/2009
• Issued: 04/03/2012
• Est. Priority Date: 06/11/2008
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Litosh, Vladislav A., Wu, Weidong, Metzker, Michael L., Hersh, Megan N., Stupi, Brian P.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/483,080
• Publication Number:
  US 20100041041A1
• Time in Patent Office: 1,027 Days
• Field of Search: 536/4.1, 435/6, 435/91.1
• US Class Current: 536/4.1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The present invention relates generally to labeled and unlabled cleavable terminating groups and methods for DNA sequencing and other types of DNA analysis. More particularly, the invention relates in part to nucleotides and nucleosides with chemically cleavable, photocleavable, enzymatically cleavable, or non-photocleavable groups and methods for their use in DNA sequencing and its application in biomedical research.

Claims

1. A compound of formula:

2. The compound of claim 1, wherein Z is —

O—

.

3. The compound of claim 1, wherein R₂ is hydrogen.

4. The compound of claim 1, wherein R₃ is hydrogen.

5. The compound of claim 1, wherein R₃ is alkyl_(C≦

8).

6. The compound of claim 5, wherein R₃ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

7. The compound of claim 1, wherein R₄ is hydrogen.

8. The compound of claim 1, wherein R₅ is hydrogen.

9. The compound of claim 1, wherein R₅ is cyano.

10. The compound of claim 1, wherein R₆ is hydrogen.

11. The compound of claim 1, wherein R₆ is a group of formula:

12. The compound of claim 11, wherein X is alkynediyl_(C2-8).

13. The compound of claim 12, wherein X is —

C═

C—

.

14. The compound of claim 11, wherein n is zero.

15. The compound of claim 1, wherein R₆ is a group of formula:

16. The compound of claim 15, wherein X is alkynediyl_(C2-8).

17. The compound of claim 16, wherein X is —

C═

C—

.

18. The compound of claim 15, wherein Y is —

CH₂—

.

19. The compound of claim 15, wherein n is zero.

20. The compound of claim 15, wherein m is zero.

21. The compound of claim 1, wherein R₆ is a -linker-reporter.

22. The compound of claim 21, wherein the linker is:

23. The compound of claim 22, wherein X is alkynediyl_(C2-8).

24. The compound of claim 23, wherein X is —

C≡

C—

.

25. The compound of claim 22, wherein n is zero.

26. The compound of claim 21, wherein the linker is:

27. The compound of claim 26, wherein X is alkynediyl_(C2-8).

28. The compound of claim 27, wherein X is —

C≡

C—

.

29. The compound of claim 26, wherein Y is —

CH₂—

.

30. The compound of claim 26, wherein n is zero.

31. The compound of claim 26, wherein m is zero.

32. The compound of claim 21, wherein the linker is:

33. The compound of claim 32, wherein X is alkynediyl_(C2-8).

34. The compound of claim 33, wherein X is —

C═

C—

.

35. The compound of claim 32, wherein Y is —

CH₂—

.

36. The compound of claim 32, wherein n is zero.

37. The compound of claim 32, wherein m is zero.

38. The compound of claim 21, wherein the reporter is based on a dye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXA FLUOR®

350, ALEXA FLUOR®

405, ALEXA FLUOR®

430, ALEXA FLUOR®

488, ALEXA FLUOR®

514, ALEXA FLUOR®

532, ALEXA FLUOR®

546, ALEXA FLUOR®

555, ALEXA FLUOR®

568, ALEXA FLUOR®

568, ALEXA FLUOR®

594, ALEXA FLUOR®

610, ALEXA FLUOR®

633, ALEXA FLUOR®

647, ALEXA FLUOR®

660, ALEXA FLUOR®

680, ALEXA FLUOR®

700, ALEXA FLUOR®

750, or a squaraine dye.

39. The compound of claim 21, wherein the reporter is:

40. The compound of claim 1, wherein R₇ is hydrogen.

41. The compound of claim 1, wherein R₈ is hydrogen.

42. The compound of claim 1, wherein R₈ is nitro.

43. The compound of claim 1, wherein R₈ is alkyl_(C≦

8).

44. The compound of claim 43, wherein R₈ is methyl.

45. The compound of claim 1, wherein R₉ is hydrogen.

46. The compound of claim 1, wherein R₉ is nitro.

47. The compound of claim 1, wherein R₉ is alkyl_(C≦

8).

48. The compound of claim 47, wherein R₉ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

49. The compound of claim 1, wherein R₉ is aryl_(C≦

8).

50. The compound of claim 49, wherein R₉ is phenyl.

51. The compound of claim 1, further defined as:

52. The compound of claim 1, further defined as:

53. The compound of claim 52, further defined as:

54. The compound of claim 52, further defined as:

55. The compound of claim 1, further defined as:

56. The compound of claim 55, further defined as:

57. The compound of claim 55, further defined as:

58. The compound of claim 1, further defined as:

59. The compound of claim 58, further defined as:

60. The compound of claim 58, further defined as:

61. The compound of claim 1, further defined as:

62. The compound of claim 61, further defined as:

63. The compound of claim 61, further defined as:

64. The compound of claim 1, further defined as:

65. The compound of claim 64, further defined as:

66. The compound of claim 64, further defined as:

67. The compound of claim 1, further defined as:

68. The compound of claim 1, further defined as:

69. The compound of claim 68, further defined as:

70. The compound of claim 68, further defined as:

71. The compound of claim 1, further defined as:

72. The compound of claim 71, further defined as:

73. The compound of claim 71, further defined as: