Methods and compositions for correcting misincorporation in a nucleic acid synthesis reaction

• US 20080096765A1
• Filed: 10/31/2007
• Published: 04/24/2008
• Est. Priority Date: 01/05/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Philip
• Application Number:
  US11/930,323
• Publication Number:
  US 20080096765A1
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2527/149 Concentration of an enzyme

Abstract

The invention provides methods for correcting misincorporation of a nucleotide in a primer during a sequencing-by-synthesis reaction by using both a polymerase substantially lacking in exonuclease activity and an enzyme, preferably a polymerase, having exonuclease activity.

Claims

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

(a) obtaining a nucleic acid duplex comprising a template and a primer hybridized thereto;

(b) exposing said duplex to a plurality of deoxynucleotide triphosphates, at least one of which is complementary to a nucleotide at a position in said template immediately adjacent to the 3′

terminus of said primer, in the presence of a first polymerase lacking exonuclease activity and a second polymerase having exonuclease activity;

(c) identifying said deoxynucleotide triphosphate that is complementary to said nucleotide; and

(d) repeating steps (b) and (c).

2. The method of claim 1, wherein said second polymerase is present in an amount sufficient to remove misincorporated deoxynucleotide triphosphate at said position.

3. The method of claim 2, wherein said second polymerase is present in an amount insufficient to remove a substantial amount of said complementary deoxynucleotide triphosphate.

4. The method of claim 2, wherein the amount of second polymerase is present as about one percent or less of the amount of first polymerase.

5. The method of claim 4, wherein the amount of second polymerase is present as less than about 0.5% of the amount of first polymerase.

6. The method of claim 4, wherein the amount of second polymerase is present as less than about 0.1% of the amount of first polymerase.

7. The method of claim 1, wherein said exposing step comprises sequentially exposing said duplex to members of said plurality.

8. The method of claim 1, wherein said exposing step comprises simultaneously exposing said duplex to members of said plurality.

9. The method of claim 1, wherein the amount of second polymerase is determined based upon the misincorporation rate of said complementary deoxynucleotide triphosphate.

10. The method of claim 1, wherein the amount of second polymerase is determined based upon the identity of the nucleotide immediately preceding said complementary deoxynucleotide triphosphate.

11. The method of claim 1, further comprising the step of compiling a sequence of deoxynucleotide triphosphates incorporated into said primer.

12. The method of claim 1, wherein said members of said plurality of deoxyribonucleotide triphosphates are detectably labeled.

13. The method of claim 12, wherein each of said members contains the same detectable label.

14. The method of claim 12, wherein each of said plurality comprises a mixture of adenosine triphosphate, guanidine triphosphate, cytidine triphosphate, and uridine triphosphate.

15. The method of claim 1, wherein said first polymerase and said second polymerase are exposed to said duplex sequentially in separate steps.

16. The method of claim 15, wherein said second polymerase is exposed to said duplex for a shorter period of time than said first polymerase.

17. The method of claim 16 wherein said second polymerase is exposed in equimolar concentration with respect to said first polymerase.