Thermus oshimai nucleic acid polymerases

• US 20030194726A1
• Filed: 11/22/2002
• Published: 10/16/2003
• Est. Priority Date: 11/30/2001
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rozzelle, James E., Bolchakova, Elena V.
• Application Number:
  US10/303,109
• Publication Number:
  US 20030194726A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention provides nucleic acids and polypeptides for a nucleic acid polymerase enzyme from a thermophilic organism, Thermus oshimai. The invention also provides methods for using these nucleic acids and polypeptides.

Claims

1. 1. An isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

2. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that decreases 5-3′

exonuclease activity.

3. The isolated nucleic acid of claim 2, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO;

5.

4. An isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

5. The isolated nucleic acid of claim 5, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5.

6. An isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

7. An isolated nucleic acid comprising a sequence complementary to SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

8. A vector comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

9. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that decreases 5-3′

exonuclease activity.

10. The vector of claim 9, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO;

5.

11. A vector comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

12. The vector of claim 11, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5.

13. A vector comprising an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

14. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

15. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that decreases 5-3′

exonuclease activity.

16. The expression vector of claim 15, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO;

5.

17. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

18. The expression vector of claim 17, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5.

19. An expression vector comprising a promoter operably linked to an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

20. The host cell of claim 19, wherein the host cell can express a thermostable polypeptide encoded by the nucleic acid, and the polypeptide has DNA polymerase activity.

21. A host cell comprising an isolated nucleic acid encoding a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

22. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that decreases 5-3′

exonuclease activity.

23. The host cell of claim 22, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO;

5.

24. A host cell comprising an isolated nucleic acid encoding a derivative nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

25. The host cell of claim 24, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5.

26. A host cell comprising an isolated nucleic acid encoding a nucleic polymerase comprising SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

27. An isolated nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

28. An isolated derivative nucleic acid polymerase comprising SEQ ID NO:

5 having a mutation that decreases 5-3′

exonuclease activity.

29. The isolated derivative nucleic acid polymerase of claim 28, wherein the derivative nucleic acid polymerase has decreased 5-3′

exonuclease activity relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO;

5.

30. An isolated derivative nucleic acid polymerase comprising SEQ ID NO:

5 having a mutation that reduces discrimination against dideoxynucleotide triphosphates.

31. The isolated derivative nucleic acid polymerase of claim 30, wherein the derivative nucleic acid polymerase has reduced discrimination against dideoxynucleotide triphosphates relative to a nucleic acid polymerase comprising amino acid sequence SEQ ID NO:

5.

32. A kit comprising a container containing a nucleic acid polymerase, wherein the nucleic acid polymerase comprises an amino acid sequence SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8.

33. The kit of claim 32 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.

34. The kit of claim 32, adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension reactions.

35. A method of making a nucleic acid polymerase comprising SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8 that comprises, incubating under conditions sufficient for RNA transcription and translation a host cell comprising a nucleic acid encoding a polypeptide comprising SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8 operably linked to a promoter.

36. The method of claim 35 wherein the nucleic acid comprises SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, or SEQ ID NO;

4.

37. A nucleic acid polymerase made by the method of claim 35.

38. A method of synthesizing DNA comprising contacting a polypeptide comprising SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8 with a DNA under conditions sufficient to permit polymerization of DNA.

39. A method for thermocyclic amplification of nucleic acid comprising:

(a) contacting a nucleic acid with a thermostable polypeptide having SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8 under conditions suitable for amplification of said nucleic acid; and

(b) amplifying the nucleic acid.

40. The method of claim 39 wherein the thermocyclic amplification of the nucleic acid includes cycles of denaturation, primer annealing and primer extension.

41. The method of claim 39 wherein the thermocyclic amplification of the nucleic acid is performed by Strand Displacement Amplification.

42. The method of claim 39 wherein the thermocyclic amplification of DNA is performed by Polymerase Chain Reaction.

43. A method of primer extending DNA comprising contacting a polypeptide comprising SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

8 with a nucleic acid under conditions sufficient to permit polymerization of DNA.

44. The method of claim 43 wherein the nucleic acid is DNA.

45. The method of claim 43 wherein the primer extension is done to sequence the nucleic acid.

46. The method of claim 43 wherein the primer extension is done to amplify the nucleic acid.