POLYMERASES AND METHODS OF USE THEREOF

• US 20100203524A1
• Filed: 10/30/2009
• Published: 08/12/2010
• Est. Priority Date: 10/31/2008
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Bowers, Jayson L., Efcavitch, J. William, Buzby, Philip R., Thompson, John F.
• Application Number:
  US12/609,543
• Publication Number:
  US 20100203524A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention generally relates to polymerases for efficient and controlled sequencing-by-synthesis reactions. In certain embodiments, the invention provides a polymerase enzyme including at least one mutation that enhances ability of the polymerase as compared to a wild-type polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA including at least one modified nucleotide.

Claims

1. A polymerase enzyme comprising at least one mutation that enhances ability of the polymerase as compared to a wild-type polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA comprising at least one modified nucleotide.

2. The polymerase according to claim 1, wherein the polymerase is a DNA polymerase.

3. The polymerase according to claim 1, wherein the modified nucleotide comprises a modification in the base portion of the nucleotide.

4. The polymerase according to claim 3, wherein the modified nucleotide comprises a residue of a cleavable linker attaching a detectable labeled to a nitrogenous base portion of the nucleotide.

5. The polymerase according to claim 4, wherein the detectable label is a fluorescent label.

6. The polymerase according to claim 5, wherein the fluorescent label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.

7. The polymerase according to claim 1, wherein the polymerase is a Taq polymerase comprising mutations that enhance ability of the polymerase to incorporate a nucleotide into a nascent strand of DNA or cDNA comprising at least one modified nucleotide compared to a wild-type Taq polymerase.

8. The polymerase according to claim 7, wherein the polymerase comprises the following mutations in the Taq polymerase:

H784Q and T664A.

9. The polymerase according to claim 7, wherein the polymerase comprises the following mutations in the Taq polymerase:

F598I, I614F, V618I, L619M, I638V, T640A, A643G, M646V, A661T, T664V, I665V, L670M, A691V, F700Y, I753V, T756S, A757G, H784Q.

10. The polymerase according to claim 7, wherein the polymerase comprises the following mutations in the Taq polymerase:

V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.

11. The polymerase according to claim 7, wherein the polymerase comprises the following mutations in the Taq polymerase:

I614F, L619M, L622F, T640E, M678K, T684A, M751T, V753I, T756S, A757G, L760I.

12. A Taq polymerase comprising the following mutations:

V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.

13. A Taq polymerase comprising the following mutations:

I614F, L619M, L622F, T640E, M678K, T684A, M751T, V753I, T756S, A757G, L760I.

14. A Taq polymerase comprising the following mutations:

V618I, L619M, V631A, I638V, T640K, M646V, M658L, A661I, T664A, I665V, L670M, F700Y, A757G, H784Q.

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

directly or indirectly anchoring a nucleic acid duplex to a surface, the duplex comprising a primer portion and a template portion having at least one modified nucleotide at its 3′

terminus;

exposing the duplex to at least one detectably labeled nucleotide in the presence of a modified polymerase capable of catalyzing addition of the nucleotide to the duplex;

detecting incorporation of the nucleotide into the primer portion; and

repeating the exposing and detecting steps at least once.

16. The method according to claim 15, further comprising:

determining a sequence of the template based upon the order of incorporation of the labeled nucleotides.

17. The method according to claim 15, further comprising:

removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.

18. The method according to claim 15, further comprising:

neutralizing the label on the labeled nucleotide after the detecting step.

19. The method according to claim 15, wherein the template is individually optically resolvable.

20. The method according to claim 15, wherein the template is attached to the surface.

21. The method according to claim 15, wherein the nucleotide is a non-native nucleotide.

22. A method for increasing the accuracy of sequencing a nucleic acid, the method comprising:

contacting a nucleic acid duplex comprising a primer nucleic acid hybridized to a template nucleic acid with a polymerase enzyme in the presence of a first labeled nucleotide under conditions that permit the polymerase to add nucleotides to said primer in a template-dependent manner, wherein the polymerase has reduced or eliminated exonuclease activity or has reduced or eliminated binding affinity of the 3′

-5′

exonuclease domain;

detecting a signal from the incorporated labeled nucleotide; and

sequentially repeating said contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of the nucleic acid.

23. The method according to claim 22, wherein said duplex is attached to a surface.

24. The method according to claim 23, wherein said surface comprises a plurality of duplex immobilized at different positions on the substrate.

25. The method according to claim 24, wherein at least some of said duplex are individually optically resolvable.

26. The method according to claim 22, wherein the label is a fluorescent label.

27. The method according to claim 22, wherein the polymerase is Klenow fragment of E. coli Pol 1 or Stoffel fragment of Taq polymerase.

28. The method according to claim 22, wherein the polymerase comprises at least one mutation that results in the lack of exonuclease activity or the reduced binding affinity of the 3′

-5′

exonuclease domain.

29. The method according to claim 22, wherein the polymerase comprises at least one mutation that results in absence of the 5′

-3′

exonuclease activity of the polymerase.

30. The method according to claim 22, wherein the polymerase comprises at least one mutation that results in absence of the 3′

-5′

exonuclease activity of the polymerase.

31. The method according to claim 22, wherein the polymerase comprises at least one mutation that results in reduced nucleic acid binding affinity of the 3′

-5′

exonuclease domain of the polymerase.

32. The method according to claim 31, wherein the polymerase is a klenow fragment comprising the following mutations:

Leu361, Glu419, Lys422, Tyr423, Arg455, Phe473, and His660.

33. The method according to claim 32, wherein Ala is substituted at any of the positions.

34. The method according to claim 22, wherein the polymerase comprises at least one mutation that affects metal ion binding.

35. The method according to claim 34, wherein the polymerase is a Klenow fragment comprising the following mutations:

Asp355, Glu357, Asp424, and Asp501.