Cloned DNA polymerases from thermotoga and mutants thereof

• US 6,015,668 A
• Filed: 09/06/1996
• Issued: 01/18/2000
• Est. Priority Date: 09/30/1994
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K., Hughes, A. John
• Primary Examiner(s): Hendricks, Keith D.
• Application Number:
  US08/706,706
• Time in Patent Office: 1,229 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/194, 435/320.1, 435/325, 435/419, 435/252.3, 536/23.2
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'"'"'→5'"'"' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'"'"'→3'"'"' exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The DNA polymerases of the invention may be used in well-known DNA sequencing and amplification reactions.

Claims

1. A method of sequencing a DNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule;

(b) contacting said first DNA molecule with deoxyribonucleoside triphosphates, a Thermotoga neapolitana DNA polymerase, and a terminator molecule to form a mixture;

(c) incubating said mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 5'"'"' termini; and

(d) separating said synthesized DNA molecules by size so that at least a portion of the nucleotide sequence of said first DNA molecule can be determined;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; and

further wherein;

said mutant Thermologa neapolitana DNA polymerase has at least one mutation selected from the group consisting of;

(1) a first mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase;

(2) a second mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'"'"'-3'"'"' exonuclease domain of said polymerase; and

(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

2. The method of sequencing as claimed in claim 1, wherein said deoxyribonucleoside triphosphates are selected from the group consisting of:

dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

3. The method of sequencing as claimed in claim 1, wherein said terminator nucleotide is selected from the group consisting of:

ddTTP, ddATP, ddGTP, ddITP, and ddCTP.

4. The method of sequencing as claimed in claim 1, wherein said DNA polymerase comprises said third mutation.

5. The method of sequencing as claimed in claim 4, wherein said third mutation is a substitution of Phe at position 730 of said DNA polymerase with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

6. The method of sequencing as claimed in claim 5, wherein said third mutation is a Phe to Tyr substitution at position 730 of said DNA polymerase.

7. The method of sequencing as claimed in claim 4, wherein said DNA polymerase further comprises said first mutation.

8. The method of sequencing as claimed in claim 7, wherein said first mutation is an Asp to Ala substitution at position 323 of said DNA polymerase.

9. The method of sequencing as claimed in claim 7, wherein said DNA polymerase further comprises said second mutation.

10. The method of sequencing as claimed in claim 9, wherein said second mutation is the deletion of the 219 N-terminal amino acids of Thermotoga neapolitana DNA polymerase.

11. A method of amplifying a double-stranded DNA molecule comprising:

(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3'"'"'-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3'"'"'-termini of the second strand of said DNA molecule;

(b) hybridizing said primer to said first strand and said second primer to said second strand in the presence of a Thermotoga neapolitana DNA polymerase, under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;

(c) denaturing said first and third strand, and said second and fourth strands; and

(d) repeating steps (a) to (c) one or more times;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; and

further wherein;

said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of;

(1) a first mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase;

(2) a second mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'"'"'-3'"'"' exonuclease domain of said polymerase; and

(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

12. The method of amplifying as claimed in claim 11, wherein said DNA polymerase comprises said second mutation.

13. The method of amplifying as claimed in claim 12, wherein said second mutation is the deletion of the 219 N-terminal amino acids of Thermotoga neapolitana DNA polymerase.

14. The method of amplifying as claimed in claim 11, wherein said DNA polymerase further comprises said third mutation.

15. The method of amplifying as claimed in claim 14, wherein said third mutation is a substitution of Phe at position 730 of said DNA polymerase with an amino acid selected from the group consisting of Lys, Arg, His, Asp, Glu, Ala, Val, Ile, Leu, Pro, Met, Trp, Gly, Ser, Tyr, Cys, Thr, Asn, and Gln.

16. The method of amplifying as claimed in claim 15, wherein said third mutation is a Phe to Tyr substitution at position 730 of said DNA polymerase.

17. The method of amplifying as claimed in claim 11, wherein said DNA polymerase further comprises said first mutation.

18. The method of amplifying as claimed in claim 17, wherein said first mutation is an Asp to Ala substitution at position 323 of said DNA polymerase.

19. A kit for sequencing a DNA molecule comprising:

(a) a first container comprising a Thermotoga neapolitana DNA polymerase;

(b) a second container comprising one or more dideoxyribonucleoside triphosphates; and

(c) a third container comprising one or more deoxyribonucleoside triphosphates;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; and

further wherein;

said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of;

(1) a first mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase;

(2) a second mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'"'"'-3'"'"' exonuclease domain of said polymerase; and

(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

20. The kit for sequencing as claimed in claim 19, wherein said DNA polymerase comprises said third mutation.

21. The kit for sequencing as claimed in claim 20, wherein said third mutation is a Phe to Tyr substitution at position 730 of said polymerase.

22. The kit for sequencing as claimed in claim 20, wherein said DNA polymerase further comprises said first mutation.

23. The kit for sequencing as claimed in claim 22, wherein said first mutation is an Asp to Ala substitution at position 323 of said polymerase.

24. The kit for sequencing as claimed in claim 22, wherein said DNA polymerase further comprises said second mutation.

25. The kit for sequencing as claimed in claim 24, wherein said second mutation is the deletion of 219 N-terminal amino acids of Thermotoga neapolitana DNA polymerase.

26. The kit for sequencing as claimed in claim 19, wherein said kit further comprises a pyrophosphatase.

27. A kit for amplifying a DNA molecule, comprising:

(a) a first container comprising a Thermotoga neapolitana DNA polymerase; and

(b) a second container comprising one or more deoxyribonucleoside triphosphates;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; and

further wherein;

said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of;

(1) a first mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase;

(2) a second mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'"'"'-3'"'"' exonuclease domain of said polymerase; and

(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

28. The kit for amplifying as claimed in claim 27, wherein said DNA polymerase comprises said second mutation.

29. The kit for amplifying as claimed in claim 28, wherein said second mutation is the deletion of the 219 N-terminal amino acids of the Thermotoga neapolitana DNA polymerase.

30. The kit for amplifying as claimed in claim 27, wherein said DNA polymerase further comprises said third mutation.

31. The kit for amplifying as claimed in claim 30, wherein said third mutation is a Phe to Tyr substitution at position 730 of said polymerase.

32. The kit for amplifying as claimed in claim 27, wherein said DNA polymerase further comprises said first mutation.

33. The kit for amplifying as claimed in claim 32, wherein said first mutation is an Asp to Ala substitution at position 323 of said DNA polymerase.

34. The kit for amplifying as claimed in claim 27, wherein said kit further comprises a pyrophosphatase.

35. A method for synthesizing a DNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule; and

(b) incubating said DNA molecule in the presence of a Thermotoga neapolitana DNA polymerase and one or more deoxyribonucleoside triphosphates under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule; and

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity; and

further wherein;

said mutant Thermotoga neapolitana DNA polymerase has at least one mutation selected from the group consisting of;

(1) a first mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said first mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase;

(2) a second mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said second mutation is in the 5'"'"'→

3'"'"' exonuclease domain of said polymerase; and

(3) a third mutation in the O-helix of said DNA polymerase, wherein said third mutation results in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

36. The method of synthesizing a DNA molecule as claimed in claim 35, wherein said primer or one or more of said deoxyribonucleoside triphosphates are fluorescently labeled.

37. The method of synthesizing a DNA molecule as claimed in claim 35, wherein said DNA polymerase comprises said first mutation, said second mutation, and said third mutation.

38. The method of synthesizing a DNA molecule as claimed in claim 35, wherein said DNA molecule comprises said third mutation, and further wherein said third mutation is a Phe to Tyr substitution at position 730 of said DNA polymerase.

39. A mutant Thermotoga neapolitana DNA polymerase, wherein said mutation is a substitution of Arg at position 722 of said DNA polymerase with an amino acids selected from the group consisting of Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys, Tyr, Gln, Asn, Lys, and His.

40. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39, wherein said DNA polymerase further comprises a mutation that reduces, substantially reduces or eliminates 3'"'"'-5'"'"' exonuclease activity of said DNA polymerase, wherein said mutation is in the 3'"'"'-5'"'"' exonuclease domain of said polymerase.

41. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39, wherein said DNA polymerase further comprises a mutation that reduces, substantially reduces or eliminates 5'"'"'-3'"'"' exonuclease activity of said DNA polymerase, wherein said mutation is in the 5'"'"'-3'"'"' exonuclease domain of said polymerase.

42. The mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39, wherein said Arg at position 722 of said DNA polymerase is substituted with an amino acid selected from the group consisting of His, Lys, Tyr, and Ala.

43. A recombinant nucleic acid molecule encoding the mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39.

44. A host cell comprising a recombinant nucleic acid molecule encoding the mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39.

45. A method of producing a mutant Thermotoga neapolitana DNA polymerase, comprising:

(a) culturing a host cell as claimed in claim 44;

(b) expressing the mutant Thermotoga neapolitana DNA polymerase; and

(c) isolating said mutant Thermotoga neapolitana DNA polymerase.

46. A method of sequencing a DNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule;

(b) contacting said first DNA molecule with deoxyribonucleoside triphosphates, a Thermotoga neapolitana DNA polymerase, and a terminator molecule to form a mixture;

(c) incubating said mixture under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator at their 5'"'"' termini; and

(d) separating said synthesized DNA molecules by size so that at least a portion of the nucleotide sequence can be determined; and

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39 and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity.

47. The method of sequencing as claimed in claim 46, wherein said deoxyribonucleoside triphosphates are selected from the group consisting of:

dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α

-S]dATP, [α

-S]dTTP, [α

-S]dGTP, and [α

-S]dCTP.

48. The method of sequencing as claimed in claim 46, wherein said terminator nucleotide is selected from the group consisting of:

ddTTP, ddATP, ddGTP, ddITP, or ddCTP.

49. A method of amplifying a double-stranded DNA molecule comprising:

(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3'"'"'-termini of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3'"'"'-termini of the second strand of said DNA molecule;

(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of a Thermotoga neapolitana DNA polymerase, under conditions such that a third DNA molecule complementary to said first strand and a fourth DNA molecule complementary to said second strand are synthesized;

(c) denaturing said first and third strand, and said second and fourth strands; and

(d) repeating steps (a) to (c) one or more times;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39 and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity.

50. A kit for sequencing a DNA molecule comprising:

(a) a first container comprising a Thermotoga neapolitana DNA polymerase;

(b) a second container comprising one or more dideoxyribonucleoside triphosphates; and

(c) a third container comprising one or more deoxyribonucleoside triphosphates;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39 and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity.

51. The kit for sequencing as claimed in claim 50, wherein said kit further comprises a pyrophosphatase.

52. A kit for amplifying a DNA molecule, comprising:

(a) a first container comprising a Thermotoga neapolitana DNA polymerase; and

(b) a second container comprising one or more deoxyribonucleoside triphosphates;

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39 and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity.

53. The kit for amplifying as claimed in claim 52, wherein said kit further comprises a pyrophosphatase.

54. A method for synthesizing a DNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule; and

(b) incubating said DNA molecule in the presence of a Thermotoga neapolitana DNA polymerase and one or more deoxyribonucleoside triphosphates under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule; and

wherein;

said Thermotoga neapolitana DNA polymerase is selected from the group consisting of a mutant Thermotoga neapolitana DNA polymerase as claimed in claim 39 and a fragment of said mutant Thermotoga neapolitana DNA polymerase, wherein said fragment has DNA polymerase activity.

55. The method of synthesizing a DNA molecule as claimed in claim 54, wherein said primer or one or more of said deoxyribonucleoside triphosphatases are fluorescently labeled.