Mutant chimeric DNA polymerase

• US 6,228,628 B1
• Filed: 06/26/1998
• Issued: 05/08/2001
• Est. Priority Date: 07/09/1997
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Reichert, Fred Lawrence, Gelfand, David Harrow
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): HUTSON, RICHARD G
• Application Number:
  US09/105,697
• Time in Patent Office: 1,047 Days
• Field of Search: 435/194, 435/252.3, 435/254.11, 435/320.1, 435/325, 536/23.2
• US Class Current: 435/194
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Mutant, chimeric thermostable DNA polymerases are provided, along with purified DNA sequences that encode the enzymes. Also provided are methods for producing and using the enzymes.

Claims

1. A thermostable DNA polymerase consisting of an N-terminal region and a C-terminal region,

wherein said N-terminal region consists of amino acids 1 through n of a Thermus species DNA polymerase, wherein n is an amino acid corresponding to an amino acid m of a Thermatoga maritima (Tma) DNA polymerase described by SEQ ID NO:

10, wherein m is between 137 and 291;

wherein said C-terminal region consists of amino acids m+1 through 893 of said Tma DNA polymerase;

wherein said N-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces or eliminates 5′

-nuclease activity when present in said Thermus species DNA polymerase, or said C-terminal region is modified by at least one point mutation within the region that is amino acids m+1 to 291 of Tma DNA polymerase, each characterized in that it substantially reduces or eliminates 5′

-nuclease activity when present in Tma DNA polymerase;

wherein said C-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces 3′

to 5′

exonuclease activity when present in Tma DNA polymerase; and

wherein said C-terminal region is modified to contain a tyrosine at amino acid 730.

2. A thermostable DNA polymerase of claim 1, wherein said N-terminal region contains a point mutation at an amino acid position corresponding to an amino acid in Taq DNA polymerase selected from the group consisting of D18, R25, G46, D67, F73, R74, Y81, G107, E117, D119, D120, D142, D144, G187, D188, D191, and G195.

3. A thermostable DNA polymerase of claim 1, wherein said C-terminal region contains a point mutation at an amino acid position selected from the group consisting of D323, E325, L329, N385, D389, L393, Y464, and D468.

4. A thermostable DNA polymerase of claim 2, wherein said N-terminal region contains an aspartic acid at an amino acid position corresponding to amino acid G46 in Taq DNA polymerase.

5. A thermostable DNA polymerase of claim 3, wherein said C-terminal region contains a D323A or E325A mutation.

6. A thermostable DNA polymerase of claim 1, wherein said Thermus species is selected from the group consisting of Thermus aquaticus, Thermus flavus, Thermus thermophilus, Thermus species Z05, Thermus caldofilus, Thermus species sps17, Thermus filiformis.

7. A thermostable DNA polymerase of claim 6, wherein said Thermus species is Thermus aquaticus.

8. A thermostable DNA polymerase of claim 7, wherein n=190.

9. A thermostable DNA polymerase of claim 8, wherein said N-terminal region contains an G46D mutation, and wherein said C-terminal region contains a D323A mutation and a E325A mutation.

55. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 1.

56. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 2.

57. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 3.

58. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 4.

59. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 5.

60. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 6.

61. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 7.

62. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 8.

63. A kit for carrying out a primer extension reaction, comprising a thermostable DNA Polymerase of claim 9.

10. An isolated DNA that encodes a thermostable DNA polymerase, wherein said thermostable DNA polymerase consists of an N-terminal region and a C-terminal region,

wherein said N-terminal region consists of amino acids 1 through n of a Thermus species DNA polymerase, wherein n is an amino acid corresponding to an amino acid m of a Thermatoga maritima (Tma) DNA polymerase described by SEQ ID NO:

10, wherein m is between 137 and 291;

wherein said C-terminal region consists of amino acids m+1 through 893 of said Tma DNA polymerase;

wherein said N-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces or eliminates 5′

-nuclease activity when present in said Thermus species DNA polymerase, or said C-terminal region is modified by at least one point mutation within the region that is amino acids m+1 to 291 of Tma DNA polymerase, each characterized in that it substantially reduces or eliminates 5′

-nuclease activity when present in Tma DNA polymerase;

wherein said C-terminal region is modified by at least one point mutation, each characterized in that it substantially reduces 3′

to 5′

exonuclease activity when present in Tma DNA polymerase; and

wherein said C-terminal region is modified to contain a tyrosine at amino acid 730.

11. An isolated DNA of claim 10, wherein said N-terminal region contains a point mutation at an amino acid position corresponding to an amino acid in Taq DNA polymerase selected from the group consisting of D18, R25, G46, D67, F73, R74, Y81, G107, E117, D119, D120, D142, D144, G187, D188, D191, and G195.

12. An isolated DNA of claim 10, wherein said C-terminal region contains a point mutation at an amino acid position selected from the group consisting of D323, E325, L329, N385, D389, L393, Y464, and D468.

13. An isolated DNA of claim 11, wherein said N-terminal region contains an aspartic acid at an amino acid position corresponding to amino acid G46 in Taq DNA polymerase.

14. An isolated DNA of claim 12, wherein said C-terminal region contains a D323A or E325A mutation.

15. An isolated DNA of claim 10, wherein said Thermus species is selected from the group consisting of Thermus aquaticus, Thermus flavus, Thermus thermophilis, Thermus species Z05, Thermus caldofilus, Thermus species sps17, Thermus filiformis.

16. An isolated DNA of claim 15, wherein said Thermus species is Thermus aquaticus.

17. An isolated DNA of claim 16, wherein n=190.

18. An isolated DNA of claim 17, wherein said N-terminal region contains a G46D mutation, and wherein said C-terminal region contains a D323A mutation and a E325A mutation.

19. A plasmid comprising the isolated DNA of claim 10.

20. A plasmid comprising the isolated DNA of claim 11.

21. A plasmid comprising the isolated DNA of claim 12.

22. A plasmid comprising the isolated DNA of claim 13.

23. A plasmid comprising the isolated DNA of claim 14.

24. A plasmid comprising the isolated DNA of claim 15.

25. A plasmid comprising the isolated DNA of claim 16.

26. A plasmid comprising the isolated DNA of claim 17.

27. A plasmid comprising the isolated DNA of claim 18.

28. An expression vector comprising the isolated DNA of claim 19.

29. An expression vector comprising the isolated DNA of claim 20.

30. An expression vector comprising the isolated DNA of claim 21.

31. An expression vector comprising the isolated DNA of claim 22.

32. An expression vector comprising the isolated DNA of claim 23.

33. An expression vector comprising the isolated DNA of claim 24.

34. An expression vector comprising the isolated DNA of claim 25.

35. An expression vector comprising the isolated DNA of claim 26.

36. An expression vector comprising the isolated DNA of claim 27.

37. A host cell transformed with an expression vector of claim 28.

38. A host cell transformed with an expression vector of claim 29.

39. A host cell transformed with an expression vector of claim 30.

40. A host cell transformed with an expression vector of claim 31.

41. A host cell transformed with an expression vector of claim 32.

42. A host cell transformed with an expression vector of claim 33.

43. A host cell transformed with an expression vector of claim 34.

44. A host cell transformed with an expression vector of claim 35.

45. A host cell transformed with an expression vector of claim 36.

46. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 37 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

47. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 38 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

48. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 39 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

49. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 40 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

50. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 41 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

51. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 42 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

52. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 43 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

53. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 44 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.

54. A method for preparing a thermostable DNA polymerase, comprising:

(a) culturing a host cell of claim 45 under conditions which promote the expression of thermostable DNA polymerase; and

(b) isolating thermostable DNA polymerase from said host cell.