Methods for improving fidelity in a nucleic acid synthesis reaction

• US 20060263790A1
• Filed: 05/20/2005
• Published: 11/23/2006
• Est. Priority Date: 05/20/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Harris, Timothy
• Application Number:
  US11/133,675
• Publication Number:
  US 20060263790A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/161 A competitive reaction step

Abstract

The invention provides methods for nucleic acid sequence determination. Generally, the invention provides methods for improving fidelity of a nucleic acid synthesis reaction.

Claims

1. A method of sequencing a nucleic acid, the method comprising the steps of:

(a) exposing a target nucleic acid to a reaction mixture comprising a primer that is complementary to a portion of said target nucleic acid, a pre-determined ratio of labeled deoxynucleotides to chain elongation inhibitors and a polymerase capable of incorporating nucleotides into said primer in a template-dependent manner;

(b) identifying an incorporated nucleotide, and

(c) repeating said exposing and identifying steps.

2. The method of claim 1, wherein said target nucleic acid is a member of a clonal population of target nucleic acids.

3. The method of claim 1, wherein said inhibitors are labeled.

4. The method of claim 2, wherein said clonal population is attached to a surface.

5. The method of claim 4, wherein said surface is a bead.

6. The method of claim 4, wherein said surface comprises multiple different clonal populations of nucleic acids.

7. The method of claim 6, wherein said multiple different clonal populations are spatially segregated from each other.

8. The method of claim 2, wherein said identifying step comprises obtaining a consensus signal from said clonal population.

9. The method of claim 1, wherein said pre-determined ratio of labeled deoxynucleotides to chain elongation inhibitors is about 2 to 1.

10. The method of claim 1, wherein said pre-determined ratio of labeled deoxynucleotides to chain elongation inhibitors is about 99 to 1.

11. The method of claim 1, wherein said labeled deoxynucleotides are attached to an optically-detectable label.

12. The method of claim 11, wherein said labeled deoxynucleotides comprise a cleavable linker to said label.

13. The method of claim 1, wherein at said deoxynucleotides and said chain elongation inhibitors contain different labels.

14. A method of sequencing a nucleic acid in a template dependent manner, the method comprising the step of:

incorporating in a template-dependent manner at least one labeled nucleotide into a primer in the presence of a labeled chain elongation inhibitor, wherein the ratio of said labeled nucleotide to said labeled chain elongation inhibitor is from about 99 to 1 to about 999 to 1.

15. The method of claim 14, further comprising the step of repeating said incorporating step sequentially.

16. The method of claim 14, further comprising the step of identifying the incorporated nucleotides.