MUTANT DNA POLYMERASES AND USES THEROF

• US 20090191560A1
• Filed: 12/19/2008
• Published: 07/30/2009
• Est. Priority Date: 09/08/1995
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K.
• Application Number:
  US12/340,572
• Publication Number:
  US 20090191560A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The present invention relates to mutant DNA polymerases which incorporate dideoxynucleotides with about the same efficiency as deoxynucleotides. The present invention also related to mutant DNA polymerases which also have substantially reduced 5′-to-3′ exonuclease activity or 3′-to-5′ exonuclease activity. The invention also relates to DNA molecules coding for the mutant DNA polymerases, and hosts containing the DNA molecules.

Claims

1. A DNA molecule comprising a coding sequence for a mutant protein, wherein said mutant protein is a mutant DNA polymerase selected from the group consisting of:

E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, Streptococcus pneumoniae polymerase, Thermus aquaticus polymerase, Thermus flavus polymerase, Thermus thermophilus polymerase, Deinococcus radiodurans polymerase, Bacillus caldotenax polymerase, E. coli bacteriophage T5 polymerase, mycobacteriophage L5 polymerase, Thermatoga maritima polymerase, and E. coli bacteriophage SP01 polymerase, and wherein said mutant DNA polymerase comprises a substitution of Tyr for Phe at a position in said polymerase corresponding to Phe₅₇₀ of wild-type T5 polymerase.

2. The DNA molecule of claim 1, further comprising a promoter, wherein said promoter is in a position and orientation with respect to the coding sequence such that the mutant protein may be expressed in a cell under the control of said promoter.

3. The molecule of claim 2, wherein said coding sequence is heterologous to said promoter.

4. A host cell comprising the DNA molecule of claim 1.

5. The host cell of claim 4, wherein said host cell is E. coli.

6. A method for producing a protein, wherein said protein is a mutant DNA polymerase selected from the group consisting of:

E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, Streptococcus pneumoniae polymerase, Thermus aquaticus polymerase, Thermus flavus polymerase, Thermus thermophilus polymerase, Deinococcus radiodurans polymerase, Bacillus caldotentex polymerase, E coli bacteriophage T5 polymerase, Mycobacteriophage L5 polymerase, Thermatoga maritima polymerase, and E. coli bacteriophage SP01 polymerase, comprising a substitution of Tyr for Phe at a position in said polymerase corresponding to Phe₅₇₀ of wild-type T5 polymerase, said method comprising;

(a) culturing a host cell comprising the DNA molecule of claim 2, and

(b) isolating said protein from said host cell.

8. A DNA molecule as claimed in claim 1, wherein said mutant protein is a mutant T5 DNA polymerase comprising a substitution of Tyr for Phe₅₇₀ of wild-type T5 polymerase.

9. The DNA molecule of claim 8, further comprising a promoter, wherein said promoter is in a position and orientation with respect to the coding sequence such that the mutant protein may be expressed in a cell under the control of said promoter.

10. The molecule of claim 8, wherein said coding sequence is heterologous to the promoter.

11. A host cell comprising the DNA molecule of claim 8.

12. The host cell of claim 11, wherein said host cell is E. coli.

13. A method for producing a protein, wherein said protein is a mutant T5 DNA polymerase comprising a substitution of Tyr for Phe₅₇₀ of wild-type T5 polymerase, said method comprising:

(a) culturing a host cell comprising the DNA molecule of claim 9, and

(b) isolating said protein from said host cell.

15. A DNA molecule as claimed in claim 1, wherein said mutant protein is a mutant Taq DNA polymerase comprising a substitution of Tyr for Phe₆₆₇ of wild-type Taq polymerase.

16. The DNA molecule of claim 15, further comprising a promoter, wherein said promoter is in a position and orientation with respect to the coding sequence such that the mutant protein may be expressed in a cell under the control of said promoter.

17. The molecule of claim 16, wherein said coding sequence is heterologous to the promoter.

18. A host cell comprising the DNA molecule of claim 15.

19. The host cell of claim 18, wherein said host cell is E. coli.

20. A method for producing a protein, wherein said protein is a mutant Taq DNA polymerase comprising a substitution of Tyr for Phe₆₆₇ of wild-type Taq polymerase, said method comprising:

(a) culturing a host cell comprising the DNA molecule of claim 16, and

(b) isolating said protein from said host cell.

22. A DNA molecule as claimed in claim 1, wherein said mutant protein is a mutant Klenow fragment of E. coli DNA polymerase I comprising a substitution of Tyr for Phe762 of wild-type Klenow fragment DNA polymerase.

23. The DNA molecule of claim 22, further comprising a promoter, wherein said promoter is in a position and orientation with respect to the coding sequence such that the mutant protein may be expressed in a cell under the control of said promoter.

24. The molecule of claim 23, wherein said coding sequence is heterologous to the promoter.

25. A host cell comprising the DNA molecule of claim 22.

26. The host cell of claim 25, wherein said host cell is E. coli.

27. A method for producing a protein, wherein said protein is a mutant Klenow fragment of E. coli DNA polymerase I comprising a substitution of Tyr for Phe762 of wild-type Klenow fragment of E. coli DNA polymerase L said method comprising:

(a) culturing a host cell comprising the DNA molecule of claim 23, and

(b) isolating said protein from said host cell.

7. A mutant DNA polymerase selected from the group consisting of a mutant of:

E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, Streptococcus pneumoniae polymerase, Thermus aquaticus polymerase, Thermus flavus polymerase, Thermus thermophilus polymerase, Deinococcus radiodurans polymerase, Bacillus caldotenax polymerase, E. coli bacteriophage T5 polymerase, Thermatoga maritima polymerase, Mycobacteriophage L5 polymerase, and E. coli bacteriophage SP01 polymerase,

wherein said mutant DNA polymerase comprises a substitution of Tyr for Phe at a position in said polymerase corresponding to Phe₅₇₀ of wild-type T5 polymerase.

14. A mutant DNA polymerase as claimed in claim 7, wherein said mutant DNA polymerase is a mutant T5 DNA polymerase comprising a substitution of Tyr for Phe₅₇₀ of wild-type T5 DNA polymerase.

21. A mutant DNA polymerase as claimed in claim 7, wherein said mutant DNA polymerase is a mutant Taq DNA polymerase comprising a substitution of Tyr for Phe₆₆₇ of wild-type Taq DNA polymerase.

28. A mutant DNA polymerase as claimed in claim 7, wherein said mutant DNA polymerase is a mutant Klenow fragment of E. coli DNA polymerase I comprising a substitution of Tyr for Phe762 of wild-type Klenow fragment of E. coli DNA polymerase I.

29. Modified gene encoding a modified Pol I-type DNA polymerase wherein said modified gene is modified to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase reside 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.

30. The modified gene of claim 29 wherein said modified DNA polymerase has substantially reduced exonuclease activity.

31. A modified DNA polymerase encoded by the modified gene of any of claims 29 to 30.

32. The modified gene of claim 29 wherein said modified DNA polymerase is a thermostable enzyme.

33. The modified gene of claim 32 wherein said thermostable enzyme is selected from the group consisting of DNA polymerase encoded by Thermus aquaticus, Thermus thermophilis, and Thennus flavus.

34. Method for production of a modified Pol I-type DNA polymerase comprising the steps of:

providing a nucleic acid molecule encoding a DNA polymerase; and

mutagenizing said nucleic acid molecule to incorporate one or more base changes in nucleotide base sequence at a region that encodes its dNMP binding site to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in the dNMP binding site.

35. Method for determining a nucleotide base sequence of a DNA molecule comprising the steps of:

incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleoside triphosphate, a Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site compared to a naturally occurring unmodified DNA polymerase, said polymerase having sufficient DNA polymerase activity and substantially reduced exonuclease activity, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, in an incubating reaction; and

separating the DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

36. The method of claim 35 wherein said DNA polymerase is a thermostable DNA polymerase.

37. The method of claim 36 wherein said thermostable DNA polymerase is selected from the group consisting of DNA polymerase encoded by Thermus aquaticus, Thermus thermophilis, and Thermus flavus.

38. The method of claim 35 wherein said DNA polymerase incorporates dideoxynucleotides with about the same efficiency as deoxynucleotides.

39. The method of claim 35 wherein said DNA polymerase has substantially reduced exonuclease activity.

40. A kit for DNA sequencing comprising a modified Pol I-type DNA polymerase modified to include a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site of the DNA polymerase;

and a reagent necessary for said sequencing selected from the group consisting of dITP, a chain terminating agent, deaza-GTP, and a manganese-containing compound.

41. A method for sequencing a strand of DNA comprising the steps of:

providing said strand hybridized with a primer able to hybridize to said strand, to form a hybridized mixture, incubating said hybridized mixture with one or more deoxyribonucleoside triphosphates, a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each said DNA product having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.

42. Method for sequencing a nucleic acid comprising:

combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site, and at least two chain terminating agents in differing amounts, under conditions favoring extension of said oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced;

separating the nucleic acid fragments by size; and

determining nucleic acid sequence wherein said agents are differentiated from each other by intensity of a label in the primer extension products.

43. A method for labeling a 3′

end of a DNA fragment comprising incubating said DNA fragment with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site, and a labeled deoxynucleotide species under conditions in which said 3′

end of said DNA fragment is extended by said polymerase and thereby labeled by addition of said labeled deoxynucleotide to said DNA fragment.

44. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double-stranded DNA sequence to form an annealed mixture and incubating the annealed mixture with a modified Pol I-type DNA polymerase modified by having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site wherein said first and second primers anneal to opposite strands of said DNA sequence with their 3′

ends directed toward each other after annealing, and with the DNA sequence to be amplified located between the first and second annealed primers.

45. A Thermus aquaticus DNA polymerase having a tyrosine at residue ₆₆₇.

49. Recombinant nucleic acid encoding any of the DNA polymerase of claims 45 to 48.

50. Method for cycle sequencing using a DNA polymerase of any one of claims 45-48 wherein said method comprises mixing said DNA polymerase with a DNA molecule to be sequenced, a primer, dNTPs, and at least one chain terminating agent to form a mixture and cycling temperature of the mixture to allow alternative extension of the primer and denaturation of the primer and DNA molecule.

46. An E. coli DNA polymerase I having a tyrosine at residue 762.

47. A purified Pol I-type DNA polymerase having a tyrosine residue at an amino acid position corresponding to E coli DNA polymerase residue 762 in its dNMP binding site provided that said polymerase is not a T7 DNA polymerase.

48. The Pol I-type DNA polymerase of claim 47 wherein its dNMP binding site comprises the amino acid sequence KN₁N₂N₃N₄N₅ N₆N₇YG/Q, wherein each N is independently any amino acid wherein the K corresponds to amino acid residue 758 of E. coli DNA polymerase I and said position N₄ is tyrosine.

51. Method for cycle sequencing, comprising step of providing an excess or equal amount of all four dNTPs compared to each of the four ddNTPs in a cycle sequencing reaction and performing said cycle sequencing reaction using a DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNTP binding site.

52. Method for cycle sequencing comprising steps of providing an effective amount of all four fluorescently labeled dideoxynucleotides than corresponding deoxynucleotides in a cycle sequencing reaction and performing said cycle sequencing reaction using a DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase residue 762 in its dNMP binding site.

53. A purified thermostable DNA polymerase having a deoxynucleotide binding site with a sequence KN₁N₂N₃N₄N₅N₆N₇YG/Q wherein each of N₁-N₃ and N₅-N₇ are independently any amino acid and N₄ is a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase I residue 762 in its dNMP binding site.

54. The purified thermostable DNA polymerase according to claim 53, wherein said DNA polymerase is not a naturally-occurring DNA polymerase.

55. The purified thermostable DNA polymerase according to claim 54, wherein said thermostable DNA polymerase is modified from a DNA polymerase selected from a group consisting of DNA polymerases from Thermus thermophilis and Thermus flavus.

56. The purified DNA polymerase according to claim 54, wherein said polymerase is modified from a Thermus aquaticus DNA polymerase to have a tyrosine residue at a position corresponding to residue 667 of a corresponding naturally-occurring unmodified polymerase.

57. The purified DNA polymerase according to claim 54, wherein said polymerase is modified to remove or reduce any exonuclease activity associated with a corresponding naturally-occurring unmodified DNA polymerase.

58. The purified DNA polymerase according to claim 56, wherein said polymerase is further modified to substantially reduce 5′

-3′

exonuclease activity.

59. Purified nucleic acid encoding a DNA polymerase according to claim 54.

60. Purified nucleic acid encoding a DNA polymerase according to claim 56.

61. A kit for DNA sequencing comprising a DNA polymerase according to claim 54 and at least one reagent selected from the group consisting of dITP, a chain terminating agent, and deaza-GTP.

62. A kit for DNA sequencing comprising a DNA polymerase according to claim 56 and at least one reagent selected from the group consisting of dITP, a chain terminating agent, and deaza-GTP.

63. A method for production of a modified DNA polymerase comprising the steps of:

providing a nucleic acid molecule encoding a thermostable DNA polymerase comprising the sequence KN₁N₂N₃N₄N₅N₆N₇YG/Q, at its dNMP binding site wherein each N is independently any amino acid and mutagenizing said nucleic acid molecule to incorporate one or more base changes in the nucleotide base sequence to encode a tyrosine residue at position N₄ corresponding to T7 DNA polymerase 526 or at E. coli DNA polymerase residue 762 in its dNMP binding site.

64. A purified Pol I-type DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E coli DNA polymerase residue 762 in its dNMP binding site.

65. The DNA polymerase according to claim 64 wherein the DNA polymerase is a Pol I-type DNA polymerase and the dNMP binding site comprises the amino acid residue sequence KN₁N₂N₃N₄N₅N₆N₇YG/Q wherein each N is independently any amino acid residue and N₄ is tyrosine.

66. The DNA polymerase according to claim 64, wherein the DNA polymerase is E. coli DNA polymerase I having a tyrosine at residue 762 or a Pol I-type DNA polymerase having a tyrosine at an amino acid position corresponding to E. coli DNA polymerase I residue 762.

67. The DNA polymerase according to claim 64 wherein said polymerase is modified to remove or reduce any exonuclease activity associated with the corresponding naturally occurring unmodified DNA polymerase.

68. Purified nucleic acid encoding a DNA polymerase according to claim 64.

69. A method for production of modified DNA polymerase having an increased ability to incorporate a dideoxynucleotide relative to a corresponding naturally occurring unmodified DNA polymerase which comprises the steps of modifying a nucleic acid molecule encoding a DNA polymerase to incorporate base changes in its nucleotide base sequence to encode a tyrosine residue at an amino acid position corresponding to T7 DNA polymerase residue 526 or at an amino acid position corresponding to E. coli DNA polymerase I residue 762 in its dNMP binding site.

70. Method for determining a nucleotide base sequence of a DNA molecule comprising steps of:

incubating a DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule in a vessel containing at least one deoxynucleotide triphosphate, a DNA polymerase having a tyrosine residue at an amino acid position corresponding to T7 DNA residue 525 or at an amino acid position corresponding to E coli DNA polymerase I residue 762 at its dNMP binding site, said polymerase having sufficient DNA polymerase activity for use in DNA sequencing and having reduced or eliminated exonuclease activity, and at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base in an incubating reaction; and

separating DNA products of the incubating reaction according to size whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined, provided that the DNA polymerase is not a T7-type DNA polymerase.

73. The method of claim 70, wherein said DNA polymerase is a Pol I-type DNA polymerase.

71. A kit for DNA sequencing comprising a DNA polymerase having a tyrosine residue at an amino acid position corresponding to E. coli DNA polymerase I residue 762, said polymerase having sufficient DNA polymerase activity for use in a DNA sequencing reaction and substantially reduced exonuclease activity, and a reagent necessary for said sequencing selected from the group consisting of dITP, a chain terminating agent, and deaza-GTP, provided that said polymerase is not a T7-type DNA polymerase.

72. The kit of claim 71, wherein said DNA polymerase is a Pol I-type DNA polymerase.