Nucleotides and nucleosides and methods for their use in DNA sequencing

• US 8,497,360 B2
• Filed: 02/28/2012
• Issued: 07/30/2013
• 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., Hersh, Megan N., Stupi, Brian P., Wu, Weidong, Metzker, Michael L.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/406,934
• Publication Number:
  US 20130035237A1
• Time in Patent Office: 518 Days
• Field of Search: 536/4.1, 435/6.1, 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, further defined as a compound of formula C.

3. The compound of claim 1, further defined as a compound of formula D.

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

O—

.

5. The compound of claim 1, wherein R₁ is a triphosphate.

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

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

8).

8. The compound of claim 7, wherein R₃ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

9. The compound claim 1, wherein R₄ is hydrogen.

10. The compound claim 1, wherein R₅ is hydrogen.

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

12. The compound of claim 11, wherein the linker is:

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

C≡

C—

.

14. The compound claim 12, wherein n is zero.

15. The compound of claim 11, 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.

16. The compound of claim 11, wherein the reporter is:

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

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

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

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

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

12) or a substituted version thereof.

22. The compound of claim 21, wherein R₈ is alkyl_(C≦

8).

23. The compound of claim 22, wherein R₈ is methyl.

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

8).

25. The compound of claim 24, wherein R₉ is methyl.

26. The compound of claim 24, wherein R₉ is tert-butyl.

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

8).

28. The compound of claim 27, wherein R₉ is phenyl.

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

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

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

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

33. A method of sequencing a target nucleic acid comprising the following steps:

(i) attaching the 5′

-end of a primer to a solid surface;

(ii) hybridizing a target nucleic acid to the primer attached to the solid surface;

(iii) adding a compound claim 11, with the proviso that where more than one type of base is present, each base is attached to a different reporter group;

(iv) adding a nucleic acid replicating enzyme to the hybridized primer/target nucleic acid complex to incorporate the composition of step (iii) into the growing primer strand, wherein the incorporated composition of step (iii) terminates the polymerase reaction at an efficiency of between about 70% to about 100%;

(v) washing the solid surface to remove unincorporated components;

(vi) detecting the incorporated reporter group to identify the incorporated composition of step (iii);

(vii) a cleavage step to remove the terminating moiety resulting in an extended primer with naturally-occurring bases;

(viii) washing the solid surface to remove the cleaved terminating group; and

(ix) repeating steps (iii) through (viii) one or more times to identify the plurality of bases in the target nucleic acid.

34. A method of sequencing a target nucleic acid comprising the following steps:

(i) attaching the 5′

-end of a target nucleic acid to a solid surface;

(ii) hybridizing a primer to the target nucleic acid attached to the solid surface;

(iii) adding a compound claim 11, with the proviso that where more than one type of base is present, each base is attached to a different reporter group;

(iv) adding a nucleic acid replicating enzyme to the hybridized primer/target nucleic acid complex to incorporate the composition of step (iii) into the growing primer strand, wherein the incorporated composition of step (iii) terminates the polymerase reaction at an efficiency of between about 70% to about 100%;

(v) washing the solid surface to remove unincorporated components;

(vi) detecting the incorporated reporter group to identify the incorporated composition of step (iii);

(vii) optionally adding one or more chemical compounds to permanently cap unextended primers;

(viii) a cleavage step to remove the terminating moiety resulting in an extended primer with naturally-occurring bases;

(ix) washing the solid surface to remove the cleaved terminating group; and

(x) repeating steps (iii) through (ix) one or more times to identify the plurality of bases in the target nucleic acid.

35. A method of performing Sanger or Sanger-type sequencing using a compound claim 11.

36. A method of performing pyrosequencing or pyrosequencing-type sequencing using a compound claim 11.