Thermus Thermophilus Nucleic Acid Polymerases

• US 20110177497A1
• Filed: 10/14/2010
• Published: 07/21/2011
• Est. Priority Date: 11/30/2001
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rozzelle, James E., BOLCHAKOVA, Elena
• Granted Patent: US 8,399,231 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The invention provides novel nucleic acid polymerases from strains GK24 and RQ-1 of Thermus thermophilus, and nucleic acids encoding those polymerases, as well as methods for using the polymerases and nucleic acids.

Claims

1. An isolated nucleic acid encoding a Thermus thermophilus strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase.

2. The isolated nucleic acid of claim 1 wherein the nucleic acid polymerase is a DNA polymerase.

3. An isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-24.

4. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that decreases 5-3′

exonuclease activity.

5. The isolated nucleic acid of claim 4, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO;

13-15.

6. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

7. The isolated nucleic acid of claim 6, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15.

8. (canceled)

9. (canceled)

10. A vector comprising an isolated nucleic acid encoding a Thermus thermophilus strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase.

11. The vector of claim 10 wherein the nucleic acid polymerase is a DNA polymerase.

12. A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-24.

13. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that decreases 5-3′

exonuclease activity.

14. The vector of claim 13, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO;

13-15.

15. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

16. The vector of claim 15, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15.

17. (canceled)

18. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a Thermus thermophilus strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase.

19. The expression vector of claim 18 wherein the nucleic acid polymerase is a DNA polymerase.

20. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-24.

21. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that decreases 5-3′

exonuclease activity.

22. The expression vector of claim 21, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO;

13-15.

23. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

24. The expression vector of claim 23, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15.

25. (canceled)

26. A host cell comprising an isolated nucleic acid encoding a Thermus thermophilus strain RQ-1 (DSM catalog number 9247) nucleic acid polymerase.

27. The host cell of claim 26 wherein the nucleic acid polymerase is a DNA polymerase.

28. A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-24.

29. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that decreases 5-3′

exonuclease activity.

30. The host cell of claim 29, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO;

13-15.

31. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

32. The host cell of claim 31, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15.

40. The isolated nucleic acid polymerase of claim 31, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15.

33. (canceled)

34. An isolated nucleic acid polymerase from Thermus thermophilus strain RQ-1 (DSM catalog number 9247).

35. The isolated nucleic acid polymerase of claim 34 wherein the nucleic acid polymerase is a DNA polymerase.

36. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-24.

37. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that decreases 5-3′

exonuclease activity.

38. The isolated nucleic acid polymerase of claim 37, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO;

13-15.

39. An isolated nucleic acid polymerase comprising any one of amino acid sequences SEQ ID NO:

13-15 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

41. A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises any one of amino acid sequences SEQ ID NO:

13-24.

42. The kit of claim 41 further comprising a container containing an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer.

43. The kit of claim 41, adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension.

44. A method of making a nucleic acid polymerase comprising any one of SEQ ID NO:

13-24 that comprises, incubating under conditions sufficient for RNA transcription and translation a host cell comprising a nucleic acid encoding a polypeptide comprising any one of SEQ ID NO;

13-24 operably linked to a promoter.

45. The method of claim 44 wherein the nucleic acid comprises any one SEQ ID NO:

1-12.

46. A nucleic acid polymerase made by the method of claim 44.

47-55. -55. (canceled)