Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

• US 9,528,151 B2
• Filed: 04/01/2014
• Issued: 12/27/2016
• Est. Priority Date: 12/01/2006
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Kim, Dae Hyun, Bi, Lanrong, Meng, Qinglin, Li, Xiaoxu
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/242,487
• Publication Number:
  US 20140315191A1
• Time in Patent Office: 1,001 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

This invention provides a process for sequencing single-stranded DNA employing modified nucleotides.

Claims

1. A method, comprising:

a) providing a plurality of primer strands bound to nucleic acid templates, a fraction of said plurality of primer strands comprising a free 3′

-OH and another fraction of said plurality of primer strands comprising an incorporated labeled nucleotide analogue reversible terminator, wherein each reversible terminator comprises a cleavable moiety bound to the 3-oxygen of the deoxyribose, wherein a label is bound via a chemically cleavable linker; and

b) extending with a polymerase one or more of said primer strands that has a free 3′

-OH with an unlabeled nucleotide analogue reversible terminator, wherein each unlabeled analogue comprises a cleavable moiety bound to the 3′

-oxygen of the deoxyribose.

2. The method of claim 1, further comprising the step of detecting the incorporated labeled reversible terminator.

3. The method of claim 2, further comprising the step of chemically cleaving the chemically cleavable linker of said incorporated labeled reversible terminator so as to remove the label.

4. The method of claim 1, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose in said incorporated labeled reversible terminator is chemically cleavable.

5. The method of claim 1, wherein the moiety cleavably linked to the 3′

-oxygen of the deoxyribose in said incorporated labeled reversible terminator is photocleavable.

6. The method of claim 1, wherein the cleavable moiety bound to the 3′

-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator is an allyl moiety or a 2-nitrobenzyl moiety.

7. The method of claim 1, wherein the cleavable moiety bound to the 3′

-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator is an allyl moiety or a 2-nitrobenzyl moiety.

8. The method of claim 1, wherein the cleavable moiety attached to the 3′

-oxygen of the deoxyribose of the incorporated labeled nucleotide analogue reversible terminator is chemically cleavable with Na₂PdCl₄/P(PhSO₃Na)₃.

9. The method of claim 1, wherein the cleavable moiety attached to the 3′

-oxygen of the deoxyribose of the unlabeled nucleotide analogue reversible terminator is chemically cleavable with Na₂PdCl₄/P(PhSO₃Na)₃.

10. The method of claim 1, wherein the label is bound by a chemically cleavable linker which is chemically cleavable with Na₂PdCl₄/P(PhSO₃Na)₃.

11. The method of claim 1, where the polymerase is a 9°

N polymerase or a variant thereof.

12. The method of claim 1, wherein the primers are self-priming moieties.

13. The method of claim 1, wherein the nucleic acid templates are bound to a solid substrate.

14. The method of claim 13, wherein the nucleic acid templates are bound to the solid substrate via 1,3-dipolar azide-alkyne cycloaddition chemistry.

15. The method of claim 13, wherein about 1000 or fewer copies of the nucleic acid template are bound to the solid substrate.

16. The method of claim 13, wherein the nucleic acid templates are bound to the solid substrate via a polyethylene glycol molecule and the solid substrate is azide-functionalized.

17. The method of claim 13, wherein the nucleic acid templates are immobilized on the solid substrate via an azido linkage, an alkynyl linkage, or biotin streptavidin interaction.

18. The method of claim 13, wherein the solid substrate is in the form of a chip, a bead, or a slide.

19. The method of claim 13, wherein the solid substrate is plastic, or glass.