One component and two component DNA Pol lll replicases and uses thereof

• US 20060211003A1
• Filed: 04/07/2005
• Published: 09/21/2006
• Est. Priority Date: 04/07/2004
• Status: Active Grant

• Current Assignee: QIAGEN North American Holdings Inc. (Qiagen NV)
• Original Assignee: Eppendorf AG
• Inventors: Peters, Lars-Erik, Ettinger, Anna
• Granted Patent: US 8,192,960 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

The invention provides one-component and two-component DNA polymerases, as well as kits comprising the same, and methods of using the same for nucleic amplification and nucleic acid sequencing.

Claims

1. A method for replicating a nucleic acid molecule, comprising subjecting a nucleic acid molecule to a replication reaction in a replication reaction mixture, which replication reaction mixture comprises a minimal functional Pol III replicase, wherein said minimal functional Pol III replicase comprises at least a first component, which first component comprises an α

subunit, wherein said minimal functional Pol III replicase lacks a clamp loader, and wherein said nucleic acid molecule is replicated in said replication reaction in said replication reaction mixture by said minimal functional Pol III.

2. The method according to claim 1, wherein said minimal functional Pol III replicase is derived from non-mesophilic bacteria.

3. The method according to claim 1, wherein said minimal functional Pol III replicase is derived from an extremophile.

4. The method according to claim 1, wherein said minimal functional Pol III replicase is derived from thermophilic bacteria.

5. The method according to claim 1, wherein said minimal functional Pol III replicase is a thermostable minimal Pol III.

6. The method according to claim 1, wherein said α

subunit is a polC α

subunit.

7. The method according to claim 1, wherein said α

subunit is a dnaE α

subunit.

8. The method according to claim 1, wherein said nucleic acid is DNA.

9. The method according to claim 8, wherein said DNA is double stranded DNA.

10. The method according to claim 1, wherein said first component further comprises an ε

subunit.

11. The method according to claim 1, wherein said minimal functional Pol III replicase is a single component Pol III replicase.

12. The method according to claim 1, wherein said minimal functional Pol III replicase is a two component Pol III replicase further comprising a second component, which second component comprises a processivity clamp.

13. The method according to claim 1, wherein said method is an amplification method, wherein said replication reaction is an amplification reaction, and said replication reaction mixture is an amplification reaction mixture.

14. The method according to claim 13, wherein said amplification method is a PCR method, wherein said amplification reaction is a polymerase chain reaction, and wherein said amplification reaction mixture is a PCR mixture.

15. The method according to claim 1, said method is a sequencing method, wherein said replication reaction is a sequencing reaction, wherein said replication reaction mixture is a sequencing reaction mixture.

16. The method according to claim 1, wherein said replication reaction mixture lacks CaCl₂.

17. The method according to claim 1, wherein said replication reaction mixture is a zwitterionic buffer comprising a weak organic acid and a weak organic base.

18. The method according to claim 1, wherein said replication reaction mixture has a pH of between about pH7.5-8.9.

19. The method according to claim 1, wherein said α

subunit is present in said replication reaction mixture at a concentration of not less than 6 ng/μ

L.

20. The method according to claim 1, wherein said nucleic acid molecule is replicated in said replication reaction in said replication reaction mixture by said minimal Pol III replicase at a rate of not less than 100 nucleotides per second.

21. A nucleic acid replication kit useful for nucleic acid replication, comprising a minimal functional Pol III replicase, wherein said minimal functional Pol III replicase comprises at least a first component, which first component comprises an α

subunit, and wherein said minimal functional Pol III replicase lacks a clamp loader.

22. The kit according to claim 21, wherein said minimal functional Pol III replicase is derived from non-mesophilic bacteria.

23. The kit according to claim 21, wherein said minimal functional Pol III replicase is derived from an extremophile.

24. The kit according to claim 21, wherein said minimal functional Pol III replicase is derived from thermophilic bacteria.

25. The kit according to claim 21, wherein said minimal functional Pol III replicase is a thermostable minimal Pol III.

26. The kit according to claim 21, wherein said α

subunit is a polC α

subunit.

27. The kit according to claim 21, wherein said α

subunit is a dnaE α

subunit.

28. The kit according to claim 21, wherein said nucleic acid is DNA.

29. The kit according to claim 28, wherein said DNA is double stranded DNA.

30. The kit according to claim 21, wherein said first component further comprises an ε

subunit.

31. The kit according to claim 21, wherein said minimal functional Pol III replicase is a single component Pol III replicase.

32. The kit according to claim 21, wherein said minimal functional Pol III replicase is a two component Pol III replicase further comprising a second component, which second component comprises a processivity clamp.

33. The kit according to claim 21, wherein said method is an amplification method, wherein said replication reaction is an amplification reaction, and said replication reaction mixture is an amplification reaction mixture.

34. The kit according to claim 21, wherein said kit is a sequencing kit useful for DNA sequencing.

35. The kit according to claim 21, wherein said kit is an amplification kit useful for DNA acid amplification.

36. The kit according to claim 35, wherein said amplification kit is a PCR kit useful for DNA amplification by PCR.

37. A nucleic acid replication reaction mixture, comprising a minimal functional Pol III replicase, wherein said minimal functional Pol III replicase comprises at least a first component, which first component comprises an α

subunit, and wherein said minimal functional Pol III replicase lacks a clamp loader.

38. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is derived from non-mesophilic bacteria.

39. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is derived from an extremophile.

40. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is derived from thermophilic bacteria.

41. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is a thermostable minimal Pol III.

42. The nucleic acid replication reaction mixture according to claim 37, wherein said α

subunit is a polC α

subunit.

43. The nucleic acid replication reaction mixture according to claim 37, wherein said α

subunit is a dnaE α

subunit.

44. The nucleic acid replication reaction mixture according to claim 37, wherein said nucleic acid is DNA.

45. The nucleic acid replication reaction mixture according to claim 44, wherein said DNA is double stranded DNA.

46. The nucleic acid replication reaction mixture according to claim 37, wherein said first component further comprises an ε

subunit.

47. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is a single component Pol III replicase.

48. The nucleic acid replication reaction mixture according to claim 37, wherein said minimal functional Pol III replicase is a two component Pol III replicase further comprising a second component, which second component comprises a processivity clamp.

49. The nucleic acid replication reaction mixture according to claim 37, wherein said replication reaction mixture is a DNA sequencing reaction mixture useful for sequencing DNA.

50. The nucleic acid replication reaction mixture according to claim 37, wherein said replication reaction mixture is a DNA amplification reaction mixture useful for amplifying DNA.

51. The nucleic acid replication reaction mixture according to claim 50, wherein said DNA amplification reaction mixture is a PCR mixture useful for amplifying DNA by PCR.

52. A nucleic acid replication reaction tube, comprising a minimal functional Pol III replicase, wherein said minimal functional Pol III replicase comprises at least a first component, which first component comprises an α

subunit, and wherein said minimal Pol III replicase lacks a clamp loader.

53. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is derived from non-mesophilic bacteria.

54. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is derived from an extremophile.

55. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is derived from thermophilic bacteria.

56. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is a thermostable minimal Pol III.

57. The nucleic acid replication reaction tube according to claim 52, wherein said α

subunit is a polC α

subunit.

58. The nucleic acid replication reaction tube according to claim 52, wherein said α

subunit is a dnaE α

subunit.

59. The nucleic acid replication reaction tube according to claim 52, wherein said nucleic acid is DNA.

60. The nucleic acid replication reaction tube according to claim 59, wherein said DNA is double stranded DNA.

61. The nucleic acid replication reaction tube according to claim 52, wherein said first component further comprises an ε

subunit.

62. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is a single component Pol III replicase.

63. The nucleic acid replication reaction tube according to claim 52, wherein said minimal functional Pol III replicase is a two component Pol III replicase further comprising a second component, which second component comprises a processivity clamp.

64. The nucleic acid replication reaction tube according to claim 52, wherein said nucleic acid replication reaction tube is a DNA sequencing reaction tube.

65. The nucleic acid replication reaction tube according to claim 52, wherein said nucleic acid replication reaction tube is a DNA amplification reaction tube.

66. The nucleic acid replication reaction tube according to claim 65, wherein said DNA amplification reaction tube is a PCR tube.

67. A reaction mixture, comprising (i) a template DNA molecule, (ii) optionally a primer hybridizable to said template DNA molecule, (iii) a minimal functional Pol III replicase, and (iv) at least one complete copy of said template DNA molecule, wherein said complete copy of said template DNA molecule is the product of a replication reaction using said minimal functional Pol III replicase, and wherein said solution lacks a clamp loader.