Nucleotides and nucleosides and methods for their use in DNA sequencing

• US 9,200,319 B2
• Filed: 10/21/2014
• Issued: 12/01/2015
• 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:
  US14/519,616
• Publication Number:
  US 20150140559A1
• Time in Patent Office: 406 Days
• Field of Search: 435/6.12, 536/22.1, 536/25.32, 536/26.1, 536/26.2
• US Class Current: 1/1
• 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 method of identifying a plurality of bases in 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 of the formula;

3. The method of claim 1, wherein the compound is incorporated by a nucleic acid replicating enzyme that is a DNA polymerase.

4. The method of claim 1, wherein the photo-cleavage is performed using a wavelength of light ranging between 300 nm to 400 nm.

5. The method of claim 1, wherein Z is —

O—

.

6. The method of claim 1, wherein R₁ is a triphosphate.

7. The method of claim 1, wherein R₁ is α

-thiotriphosphate.

8. The method of claim 1, wherein R₂ is hydrogen.

9. The method of claim 1, wherein R₃ is alkyl_(C≦

12) or a substituted version thereof.

10. The method of claim 1, wherein R₃ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl and tert-butyl.

11. The method of claim 1, wherein R₃ is tert-butyl.

12. The method of claim 1, wherein R₄ is hydrogen.

13. The method of claim 1, wherein R₅ is hydrogen.

14. The method of claim 1, wherein the linker of the -linker-reporter is:

15. The method of claim 14, wherein X is —

C≡

C—

.

16. The method of claim 14, wherein n is zero.

17. The method of claim 14, wherein the reporter is:

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

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

2. A method of identifying a plurality of bases in 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 of the formula;