High fidelity DNA polymerase compositions and uses therefor

• US 7,932,070 B2
• Filed: 08/23/2002
• Issued: 04/26/2011
• Est. Priority Date: 12/21/2001
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sorge, Joseph A., Hogrefe, Holly, Borns, Michael
• Primary Examiner(s): Hutson; Richard G
• Application Number:
  US10/227,110
• Publication Number:
  US 20030143577A1
• Time in Patent Office: 3,168 Days
• Field of Search: 530/350, 435/194, 435/183
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The subject invention relates to compositions comprising an enzyme mixture which comprises a first enzyme and a second enzyme, where the first enzyme comprises a DNA polymerization activity and the second enzyme comprises an 3′-5′ exonuclease activity and a reduced DNA polymerization activity. The invention also relates to the above compositions in kit format and methods for high fidelity DNA synthesis using the subject compositions of the invention.

Claims

1. An enzyme mixture comprising a first enzyme and a second enzyme, wherein said first enzyme comprises 5′

-3′

polymerization activity of a DNA polymerase or reverse transcriptase, and said second enzyme is a mutant of a wild type DNA polymerase, said wild type DNA polymerase comprising the partitioning domain sequence YXGG (SEQ ID NO;

6), the polymerase domain sequence DXXSLYP (SEQ ID NO;

1) or DFRALYP (SEQ ID NO;

13), the polymerase domain sequence YXDTDS (SEQ ID NO;

4), YIDTDG (SEQ ID NO;

15), YADTDG (SEQ ID NO;

16), or YSDTDG (SEQ ID NO;

17), and the polymerase domain sequence KXY, wherein said second enzyme comprises an amino acid substitution at an amino acid position corresponding to G387 of SEQ ID NO;

19, and wherein said second enzyme comprises 3′

-5′

exonuclease activity and reduced 5′

-3′

DNA polymerization activity as compared to the wild type DNA polymerase.

2. The enzyme mixture of claim 1, wherein said first enzyme is a DNA polymerase or a reverse transcriptase.

3. The enzyme mixture of claim 2, wherein said first enzyme is selected from the group consisting of:

Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, Tli DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase and DP1/DP2 DNA polymerase.

4. The enzyme mixture of claim 1, wherein said first enzyme is a Pfu DNA polymerase.

5. The enzyme mixture of claim 1, further comprising a PCR enhancing factor and/or an additive.

6. The enzyme mixture of claim 1, wherein said enzyme mixture has a ratio of polymerization activity/exonuclease activity of (2.5-5U)/(0.02-5U).

7. The enzyme mixture of claim 6, wherein said enzyme mixture has a ratio of polymerization activity/exonuclease activity of (2.5U)/(0.04-0.08U).

18. The enzyme mixture of claim 1, wherein said first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

20. The enzyme mixture of claim 1 or 4, wherein said second enzyme is a mutant DNA polymerase from the species Pyrococcus furiosis.

23. The enzyme mixture of claim 1, wherein said wild type DNA polymerase is a Family B DNA polymerase.

24. The enzyme mixture of claim 1, wherein said wild type DNA polymerase is selected from the group consisting of Thermococcus litoralis DNA polymerase (Vent DNA polymerase) having the amino acid sequence of SEQ ID NO:

49, Pyrococcus GB-D (Deep Vent) DNA polymerase having the amino acid sequence of SEQ ID NO;

59, Thermococcus gorgonarius DNA polymerase having the amino acid sequence of SEQ ID NO;

29, Pyrococcus furiousus DNA polymerase having the amino acid sequence of SEQ ID NO;

19, and Pyrococcus kodakaraensis DNA polymerase having the amino acid sequence of SEQ ID NO;

39.

25. The enzyme mixture of claim 24, wherein said second enzyme comprises an amino acid substitution at G387 in the amino acid sequence of SEQ ID NO:

19.

26. The enzyme mixture of claim 24, wherein said second enzyme comprises an amino acid substitution at G386 in the amino acid sequence of SEQ ID NO:

29.

27. The enzyme mixture of claim 24, wherein said second enzyme comprises an amino acid substitution at G386 in the amino acid sequence of SEQ ID NO:

39.

28. The enzyme mixture of claim 24, wherein said second enzyme comprises an amino acid substitution at G389 in the amino acid sequence of SEQ ID NO:

49.

29. The enzyme mixture of claim 24, wherein said second enzyme comprises an amino acid substitution at G387 in the amino acid sequence of SEQ ID NO:

59.

30. The enzyme mixture of claim 23, wherein said second enzyme comprises the amino acid sequence of SEQ ID NO:

19 except for an amino acid substitution at G387.

39. The enzyme mixture of claim 25, wherein the amino acid substitution at G3 87 is a glycine to proline substitution.

40. The enzyme mixture of claim 30, wherein the amino acid substitution at G3 87 is a glycine to proline substitution.

43. The enzyme mixture of claim 39, wherein the first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

44. The enzyme mixture of claim 40, wherein the first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

47. The enzyme mixture of claim 43, wherein the first enzyme is Taq DNA polymerase.

48. The enzyme mixture of claim 44, wherein the first enzyme is Taq DNA polymerase.

8. An enzyme mixture comprising three or more enzymes, wherein at least one enzyme in said enzyme mixture is a mutant of a wild type DNA polymerase, said wild type DNA polymerase comprising the partitioning domain sequence YXGG (SEQ ID NO:

6), the polymerase domain sequence DXXSLYP (SEQ ID NO;

1) or DFRALYP (SEQ ID NO;

13), the polymerase domain sequence YXDTDS (SEQ ID NO;

4), YIDTDG (SEQ ID NO;

15), YADTDG (SEQ ID NO;

16), or YSDTDG (SEQ ID NO;

17), and the polymerase domain sequence KXY, wherein said mutant DNA polymerase comprises 3′

-5′

exonuclease activity and a reduced DNA polymerization activity as compared to the wild type DNA polymerase, and wherein said mutant DNA polymerase comprises an amino acid substitution at an amino acid position corresponding to G387 of SEQ ID NO;

19.

9. The enzyme mixture of claim 8, wherein at least two enzymes in said mixture are mixed as an enzyme blend before being added to said enzyme mixture.

10. The enzyme mixture of claim 9, wherein said enzyme blend comprises a Pfu DNA polymerase and Taq DNA polymerase.

11. The enzyme mixture of claim 10, wherein said enzyme blend further comprises a PCR enhancing factor.

21. The enzyme mixture of claim 8, wherein said at least one enzyme is a mutant DNA polymerase from the species Pyrococcus furiosis.

12. A kit comprising a first enzyme, a second enzyme, and packaging material therefor, wherein said first enzyme is a DNA polymerase or reverse transcriptase comprising 5′

-3′

polymerization activity of a DNA polymerase or reverse transcriptase, said second enzyme is a mutant of a wild type DNA polymerase, said wild type DNA polymerase comprising the partitioning domain sequence YXGG (SEQ ID NO;

6), the polymerase domain sequence DXXSLYP (SEQ ID NO;

1) or DFRALYP (SEQ ID NO;

13), the polymerase domain sequence YXDTDS (SEQ ID NO;

4), YIDTDG (SEQ ID NO;

15), YADTDG (SEQ ID NO;

16), or YSDTDG (SEQ ID NO;

17), and the polymerase domain sequence KXY, wherein said second enzyme comprises an amino acid substitution at an amino acid position corresponding to G387 of SEQ ID NO;

19 and wherein said second enzyme comprises 3′

-5′

exonuclease activity and a reduced DNA polymerization activity as compared to the wild type DNA polymerase.

13. The kit of claim 12, wherein said first enzyme is selected from the group consisting of:

Taq DNA polymerase, Tth DNA polymerase, UlTma DNA polymerase, Tli DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase and DP1/DP2 DNA polymerase.

14. The kit of claim 12, wherein said first enzyme is a Pfu DNA polymerase.

15. The kit of claim 12 or 14, further comprising one or more components selected from the group consisting of:

a deoxynucleotide, a reaction buffer, a PCR enhancing factor and/or an additive, a control DNA template and a control primer.

16. The kit of claim 12, wherein said enzyme mixture has a ratio of polymerization activity/exonuclease activity of (2.5-5U)/(0.02-5U).

17. The kit of claim 16, wherein said enzyme mixture has a ratio of polymerization activity/exonuclease activity of (2.5U)/(0.04-0.08U).

19. The kit of claim 12, wherein said first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

22. The kit of claim 12 or 14, wherein said second enzyme is a mutant DNA polymerase from the species Pyrococcus furiosis.

31. The kit of claim 12, wherein said wild type DNA polymerase is a Family B DNA polymerase.

32. The kit of claim 12, wherein said wild type DNA polymerase is selected from the group consisting of Thermococcus litoralis DNA polymerase (Vent DNA polymerase) having the amino acid sequence of SEQ ID NO:

49, Pyrococcus GB-D (Deep Vent) DNA polymerase having the amino acid sequence of SEQ ID NO;

59, Thermococcus gorgonarius DNA polymerase having the amino acid sequence of SEQ ID NO;

29, Pyrococcus furiousus DNA polymerase having the amino acid sequence of SEQ ID NO;

19, and Pyrococcus kodakaraensis DNA polymerase having the amino acid sequence of SEQ ID NO;

39.

33. The kit of claim 32, wherein said second enzyme comprises an amino acid substitution at G387 in the amino acid sequence of SEQ ID NO:

19.

34. The kit of claim 32, wherein said second enzyme comprises an amino acid substitution at G386 in the amino acid sequence of SEQ ID NO:

29.

35. The kit of claim 32, wherein said second enzyme comprises an amino acid substitution at G386 in the amino acid sequence of SEQ ID NO:

39.

36. The kit of claim 32, wherein said second enzyme comprises an amino acid substitution at G389 in the amino acid sequence of SEQ ID NO:

49.

37. The kit of claim 32, wherein said second enzyme comprises an amino acid substitution at G387 in the amino acid sequence of SEQ ID NO:

59.

38. The kit of claim 32, wherein said second enzyme comprises the amino acid sequence of SEQ ID NO:

19 except for an amino acid substitution at G387.

41. The kit of claim 33, wherein the amino acid substitution at G387 is a glycine to proline substitution.

42. The kit of claim 38, wherein the amino acid substitution at G387 is a glycine to proline substitution.

45. The kit of claim 41, wherein the first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

46. The kit of claim 42, wherein the first enzyme is Taq DNA polymerase or Pfu DNA polymerase.

49. The kit of claim 45, wherein the first enzyme is Taq DNA polymerase.

50. The kit of claim 46, wherein the first enzyme is Taq DNA polymerase.