Nucleotide analogs

• US 20070117102A1
• Filed: 11/22/2005
• Published: 05/24/2007
• Est. Priority Date: 11/22/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Buzby, Philip
• Application Number:
  US11/286,516
• Publication Number:
  US 20070117102A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The invention provides nucleotide analogs for use in sequencing nucleic acid molecules.

Claims

1. A nucleotide analog having the structure:

2. The nucleotide analog of claim 1, wherein B is selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof.

3. The nucleotide analog of claim 1, wherein the detectable moiety D is selected from the group consisting of fluorescein, BODIPY, EDANS, rhodamine, Cy3, Cy5, and derivatives thereof.

4. The nucleotide analog of claim 1, wherein the quenching moiety Q is selected from the group consisting of fluorescein, BODIPY, EDANS, rhodamine, Cy3, Cy5, DABCYL, and derivatives thereof.

5. The nucleotide analog of claim 1, wherein the cleavable linkage X₅ is a chemically cleavable linkage.

6. The nucleotide analog of claim 1, wherein the chemically cleavable linkage is a disulfide bond.

7. The nucleotide analog of claim 1, wherein the cleavable linkage X₅ is a photochemically cleavable linkage.

8. The nucleotide analog of claim 7, wherein the photochemically cleavable linkage is selected from the group consisting of o-nitrobenzyl and derivatives thereof.

9. The nucleotide analog of claim 1, wherein the cleavable linkage is selected from the group consisting of

10. The nucleotide analog of claim 1, wherein X₁ is S.

11. The nucleotide analog of claim 1, wherein X₂ is PO₄.

12. The nucleotide analog of claim 10, wherein X₂ is PO₄.

13. A method for nucleic acid sequence determination, comprising the steps of:

a) exposing a target nucleic acid to a primer that is complementary to at least a portion of the target nucleic acid under conditions suitable for hybridizing the primer to the target nucleic acid, a nucleotide analog of claim 1, and a polymerizing agent, under conditions suitable for extending the primer in a template-dependent manner;

b) detecting incorporation of a nucleotide in each extended primer;

c) treating each hybridized extended primer of b) such that R₂ is removed; and

d) repeating steps a), b) and c), thereby determining the sequence of the target nucleic acid.

14. The method of claim 13, R₂ being removed photochemically.

15. The method of claim 13, R₂ being removed by treating the hybridized extended primer with a reducing agent.

16. The method of claim 15, the reducing agent being selected from the group consisting of dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride.

17. The method of claim 15, further comprising treating the hybridized extended primer with a capping agent.

18. The method of claim 17, the capping agent being iodoacetamide.

19. The method of claim 13, wherein X₂ is PO₄, the method further comprising treating the hybridized extended primer of b) with an enzyme to remove said PO₄, such that the extended primer can be further extended in subsequent steps.

20. The method of claim 13, wherein said target nucleic acid is attached to a substrate.

21. The method of claim 13, wherein said target nucleic acid is individually optically resolvable.