Reaction buffer composition for nucleic acid replication with packed DNA polymerases

• US 20080038782A1
• Filed: 01/08/2007
• Published: 02/14/2008
• Est. Priority Date: 01/06/2006
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Borns, Michael
• Granted Patent: US 7,939,645 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

The invention relates to compositions, methods, and kits for nucleic acid replication, including polymerase chain reaction (PCR) and mutagenesis reactions. A buffer composition is provided which allows higher concentrations of DNA polymerase to be used, resulting in greater yield of amplified product and faster reaction kinetics.

Claims

1. A composition for nucleic acid replication, comprising a mineral salt and ammonium sulfate, having a mineral salt:

ammonium sulfate molar ratio in the range from 5;

1 to 240;

1.

2. The composition of claim 1, wherein the mineral salt:

ammonium salt molar ration is in the range from 5;

1 to 80;

1.

3. The composition of claim 1, wherein the mineral salt concentration is in the range from 50 mM to 120 mM.

4. The composition of claim 2, wherein the mineral salt concentration is in the range from 80 mM to 100 mM.

5. The composition of claim 1, wherein the ammonium sulfate concentration is in the range from 1 mM to 5 mM.

6. The composition of claim 1, wherein the mineral salt concentration is in the range from 50 mM to 120 mM and the ammonium sulfate concentration is in the range from 1 mM to 5 mM.

7. The composition of claim 1, further comprising magnesium sulfate.

8. The composition of claim 7, wherein the magnesium sulfate concentration is in the range from 1 mM to 3 mM.

9. The composition of claim 8, wherein the magnesium sulfate concentration is 2 mM.

10. The composition of claim 1, further comprising Tris sulfate buffer.

11. The composition of claim 10, wherein the Tris sulfate concentration is in the range from 15 mM to 50 mM.

12. The composition of claim 11, wherein the Tris sulfate concentration is 30 mM.

13. The composition of claim 10, wherein the pH of the solution is in the range from 8 to 11.

14. The composition of claim 13, wherein the pH is 10.

15. The composition of claim 13, wherein the pH is 9.

16. The composition of claim 1, wherein the mineral salt concentration is in the range from 50 mM to 120 mM, the ammonium sulfate concentration is in the range from 1 mM to 5 mM, and comprising magnesium sulfate at a concentration in the range from 1 mM to 3 mM and Tris sulfate at a concentration in the range from 15 mM to 50 mM.

17. The composition of claim 1, comprising 50 mM mineral salt, 1 mM ammonium sulfate, 30 mM Tris sulfate adjusted to pH 10.0, 2 mM magnesium sulfate, and 0.1% Triton X-100.

18. The composition of claim 1, comprising 80 mM mineral salt, 1.5 mM ammonium sulfate, 30 mM Tris sulfate adjusted to pH 10.0, 2 mM magnesium sulfate, and 0.1% Triton X-100.

19. The composition of claim 1, comprising 100 mM mineral salt, 2 mM ammonium sulfate, 30 mM Tris sulfate adjusted to pH 10.0, 2 mM magnesium sulfate, and 0.1% Triton X-100.

20. The composition of claim 19, wherein essentially all of the anions are sulfate ions.

21. The composition of claim 1, further comprising one or more nucleotides.

22. The composition of claim 1, further comprising one or more DNA polymerases.

23. The composition of claim 22, wherein said DNA polymerase is exo- Pfu-Sso7d or exo- Pfu V93R-Sso7d.

24. The composition of claim 22, comprising a DNA polymerase at a concentration of greater than 1 unit per 50 μ

l.

25. The composition of claim 24, wherein the DNA polymerase concentration is at least 3 units per 50 μ

l.

26. The composition of claim 24, wherein the DNA polymerase concentration is at least 4 units per 50 μ

l.

27. The composition of claim 24, wherein the DNA polymerase concentration is at least 5 units per 50 μ

l.

29. The composition of claim 24, wherein the DNA polymerase concentration is at least 10 units per 50 μ

l.

30. The composition of claim 24, wherein the DNA polymerase concentration is at least 20 units per 50 μ

l.

31. The composition of claim 24, wherein the DNA polymerase concentration is in the range of 3 to 25 units per 50 μ

L.

32. The composition of claim 1, further comprising a nucleic acid template.

33. The composition of claim 1, further comprising a primer.

34. The composition of claim 1, further comprising a polymerization enhancing factor.

35. The composition of claim 1, further comprising one or more nucleotides, one or more nucleic acid polymerases, a nucleic acid template, and a primer.

36. A buffer concentrate for preparing a DNA polymerase reaction solution, wherein dilution by a factor ranging from 2-fold to 20-fold yields the composition of claim 1.

37. The buffer concentrate of claim 36, wherein dilution by a factor ranging from 5-fold to 10-fold yields the composition of claim 1.

38. The buffer concentrate of claim 36, wherein dilution by 5-fold to 10-fold yields the composition of claim 16.

39. The buffer concentrate of claim 36, wherein dilution by 5-fold to 10-fold yields the composition of claim 17.

40. The buffer concentrate of claim 36, wherein dilution by 5-fold to 10-fold yields the composition of claim 18.

41. The buffer concentrate of claim 36, further comprising one or more nucleotides.

42. The buffer concentrate of claim 36, further comprising one or more nucleic acid polymerases.

43. The buffer concentrate of claim 42, wherein the DNA polymerase is a Pfu-Sso7d or exo- Pfu V93R-Sso7d.

44. The buffer concentrate of claim 42, wherein the DNA polymerase is Pyrococcus GB-D-furiosus-Sso7d.

45. The buffer concentrate of claim 36, further comprising a Fen nuclease.

46. The buffer concentrate of claim 36, further comprising a nucleic acid template.

47. The buffer concentrate of claim 36, further comprising a primer.

48. The buffer concentrate of claim 36, further comprising a polymerization enhancing factor.

49. A method of replicating DNA, comprising performing a DNA polymerase reaction using the composition of claim 1.

50. A method of replicating a nucleic acid, comprising performing a DNA polymerase reaction, wherein the DNA polymerase reaction is performed using a dilution of the buffer concentrate of claim 36.

51. The method of claim 49, wherein said nucleic acid is amplified.

52. The method of claim 49, wherein PCR is performed.

53. The method of claim 49, wherein RT-PCR is performed.

54. The method of claim 49, wherein QPCR is performed.

55. The method of claim 49, wherein mutagenesis is performed.

56. The method of claim 55, wherein said mutagenesis is random.

57. The method of claim 55, wherein said mutagenesis is site directed.

58. The method of claim 49, wherein the concentration of DNA polymerase in said reaction is at least 2 units per 100 μ

l.

59. The method of claim 49, wherein the concentration of DNA polymerase in said reaction is at least 3 units per 100 μ

l.

60. The method of claim 49, wherein the concentration of DNA polymerase in said reaction is at least 4 units per 100 μ

l.

61. The method of claim 49, wherein the concentration of DNA polymerase in said reaction is at least 5 units per 100 μ

l.

62. The method of claim 49, wherein the concentration of DNA polymerase in said reaction is at least 6 units per 100 μ

l.

63. The method of claim 49, wherein the DNA polymerase is thermostable.

64. The method of claim 63, wherein said reaction is performed at a temperature of at least 70°

C.

65. The method of claim 63, wherein said reaction is performed at a temperature of at least 90°

C.

66. The method of claim 49, wherein the DNA polymerase is Pfu or Taq polymerase.

67. A kit for the replication of a nucleic acid sample, comprising the buffer concentrate of claim 41 and packaging material therefor.

68. The kit of claim 67 further comprising one or more nucleotides, one or more nucleic acid polymerases, a Fen nuclease, one or more primers, a nucleic acid template, or a polymerization enhancing factor.

69. The composition of claim 1, wherein the mineral salt is selected from the group consisting of potassium sulfate, potassium chloride, potassium nitrate, cesium chloride, and cesium nitrate.

70. The composition of claim 69, wherein the mineral salt is potassium sulfate.

71. The composition of claim 1, wherein the mineral salt is a mixture of salts selected from the group consisting of potassium sulfate, potassium chloride, potassium nitrate, cesium chloride, and cesium nitrate.

72. The composition of claim 1, wherein the cation concentration of the mineral salt component of said composition is in the range of 50 mM and 120 mM.

73. The composition of claim 22, wherein the DNA polymerase is Pfu-Deep Vent-Sso7d.

28. The composition of claim24, wherein the DNA polymerase concentration is at least 6 units per 50 μ

l.