Fluorescently labeled nucleoside triphosphates and analogs thereof for sequencing nucleic acids

• US 20050170367A1
• Filed: 06/10/2004
• Published: 08/04/2005
• Est. Priority Date: 06/10/2003
• Status: Abandoned Application

• Current Assignee: California Institute of Technology, Helicos Biosciences Corporation
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Buzby, Philip R.
• Application Number:
  US10/866,388
• Publication Number:
  US 20050170367A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides

Abstract

The invention provides methods for sequencing a nucleic acid, and particularly methods for synthesizing fluorescently labeled nucleoside triphosphates and related analogs for sequencing nucleic acids.

Claims

1. A fluorescently labeled nucleoside triphosphate comprising the structure:

Triphosphate-R₁R₂R₃R₄R₅-Fluorescent Label

wherein when R₁ is deoxyribose

2. The fluorescently labeled nucleoside triphosphate of claim 1, wherein R₃ is propyne.

3. The fluorescently labeled nucleoside triphosphate of claim 1, wherein R₄ is a sulfide group.

4. The fluorescently labeled nucleoside triphosphate of claim 1, wherein R₅ is a cleavable linker that is chemically cleaved.

5. The fluorescently labeled nucleoside triphosphate of claim 4, wherein R₅ is a chemically cleavable linker that is an amino acid or a hydroxyl acid derivative.

6. The fluorescently labeled nucleoside triphosphate of claim 4, wherein R₅ is a chemically cleavable linker selected from the group consisting of

7. The fluorescently labeled nucleoside triphosphate of claim 4, wherein R₅ is a chemically cleavable linker cleaved under acidic, basic, oxidative, reductive or aqueous ring closing metathesis conditions.

8. The fluorescently labeled nucleoside triphosphate of claim 1, wherein R₅ is an extended linker that has a carboxyl acid functionality and a heteroatom.

9. The fluorescently labeled nucleoside triphosphate of claim 8, wherein the extended linker has the following structure:

10. The fluorescently labeled nucleoside triphosphate of claim 9, wherein the extended linker is 6-aminohexanoic acid.

11. A method for determining a nucleic acid sequence, the method comprising the step of incorporating the fluorescently labeled nucleoside triphosphate of claim 1 to a nucleic acid.

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

(a) exposing a target nucleic acid to a primer that is complementary to at least a portion of the target, a fluorescently labeled nucleoside triphosphate of claim 1, and a polymerizing agent;

(b) conducting a primer extension;

(c) detecting incorporation of said nucleoside in said primer; and

,

(d) repeating steps (a), (b) and (c), thereby to determine a sequence of said target.

13. The method of claim 12, further comprising the step of cleaving the fluorescently labeled nucleoside triphosphate.

14. The method of claim 13, wherein the cleavage step is performed by using photolysis or chemical hydrolysis.

15. The method of claim 12, wherein the fluorescently labeled nucleoside triphosphate lacks a 3′

hydroxyl group.

16. The method of claim 12, wherein the fluorescently labeled nucleoside triphosphate label comprises a label selected from the group consisting of cyanine, rhodamine, fluorescein, coumarin, BODIPY, alexa, or conjugated multi-dyes.

17. The method of claim 12, wherein said target is attached to a substrate.

18. The method of claim 12, further comprising the step of washing an unincorporated nucleoside or analog thereof.

19. The method of claim 12, further comprising the step of compiling a sequence of said target based upon said complement sequence.

20. The method of claim 12, wherein said detecting step comprises detecting coincident fluorescence emission of a first fluorescent label and a second fluorescent label.

21. The method of claim 20, wherein the coincident fluorescence emission spectrum is between about 400 nm to about 900 nm.

22. The method of claim 21, wherein said coincident detection represents the presence of a single labeled molecule.

23. The method of claim 12, wherein said fluorescently labeled nucleoside triphosphate is a non-chain terminating nucleotide.

24. The method of claim 23, wherein said non-chain terminating nucleotide is a deoxynucleotide selected from the group consisting of dATP, dTTP, dUTP, dCTP, and dGTP.

25. The method of claim 23, wherein said non-chain terminating nucleotide is a ribonucleotide selected from the group consisting of ATP, UTP, CTP, and GTP.