Cloned DNA polymerases from Thermotoga neapolitana

• US 5,912,155 A
• Filed: 01/09/1995
• Issued: 06/15/1999
• 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 Jr.
• Primary Examiner(s): Hendricks, Keith D.
• Application Number:
  US08/370,190
• Time in Patent Office: 1,618 Days
• Field of Search: 435/194, 435/320.1, 435/252.3, 435/252.33, 536/23.2
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• 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: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• 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 neapolitana (Tne). The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The present invention also relates to the cloning and expression of the Tne DNA polymerase in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The Tne DNA polymerase of the invention may be used in well-known DNA sequencing and amplification reactions.

Claims

1. A substantially pure thermostable Thermotoga neapolitana DNA polymerase, wherein said DNA polymerase is a Pol I-type polymerase.

2. The substantially pure thermostable Thermotoga neapolitana DNA polymerase as claimed in claim 1, wherein said polymerase has a molecular weight of about 100 kDa.

3. The substantially pure thermostable Thermotoga neapolitana DNA polymerase as claimed in claim 1, wherein said DNA polymerase has the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

4. A substantially pure Thermotoga neapolitana DNA polymerase having a molecular weight of about 100 kDa, or fragments thereof having DNA polymerase activity.

5. The substantially pure Thermotoga neapolitana DNA polymerase or fragments as claimed in claim 4, wherein said DNA polymerase has the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

6. The substantially pure Thermotoga neapolitana DNA polymerase or fragments thereof as claimed in claim 4, wherein said DNA polymerase comprises the amino acid sequence of SEQ ID NO:

3.

7. The substantially pure Thermotoga neapolitana DNA polymerase or fragments thereof as claimed in claim 4, wherein said DNA polymerase has reverse transcriptase activity.

8. The substantially pure Thermotoga neapolitana DNA polymerase or fragments thereof as claimed in claim 4, wherein said DNA polymerase has an enhanced rate of incorporation of dATP relative to ³⁵ S! Thermus aguaticus DNA polymerase.

9. A substantially pure Thermotoga neapolitana DNA polymerase, wherein said polymerase is encoded by a DNA molecule having the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4.

10. A fragment of a substantially pure Thermotoga neapolitana DNA polymerase, wherein said polymerase is encoded by a DNA molecule having the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4 and said fragment has DNA polymerase activity.

11. The fragment of a substantially pure Thermotoga neapolitana DNA polymerase as claimed in claim 10, wherein said fragment has reverse transcriptase activity.

12. The fragment of a substantially pure Thermotoga neapolitana DNA polymerase as claimed in claim 10, wherein said fragment has an enhanced rate of incorporation of ³⁵ S! dATP relative to Thermus aquaticus DNA polymerase.

13. An isolated nucleic acid molecule encoding a thermostable Thermotoga neapolitana DNA polymerase, wherein said DNA polymerase is a Pol I-type polymerase.

14. The isolated nucleic acid molecule as claimed in claim 13, wherein said nucleic acid molecule encodes a Thermotoga neapolitana DNA polymerase having a molecular weight of about 100 kDa.

15. The isolated nucleic acid molecule as claimed in claim 13, wherein said DNA polymerase has the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

26. A recombinant nucleic acid molecule, wherein said recombinant nucleic acid molecule comprises the polynucleotide sequence of an isolated nucleic acid molecule as claimed in any one of claims 13, 16, 22, and 23.

27. The isolated nucleic acid molecule as claimed in any one of claims 13, 16, 22, and 23, wherein said nucleic acid molecule is a DNA molecule.

28. The isolated nucleic acid molecule as claimed in claim 27, wherein said DNA molecule further comprises a promoter.

29. The isolated nucleic acid molecule as claimed in claim 28, wherein said promoter is an inducible promoter selected from the group consisting of:

a tac promoter, a trp promoter, and a trc promoter.

16. An isolated nucleic acid molecule encoding a Thermotoga neapolitana DNA polymerase having a molecular weight of 100 kDa, or fragments thereof having DNA polymerase activity.

17. The isolated nucleic acid molecule as claimed in claim 16, wherein said DNA polymerase has the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

18. The isolated nucleic acid molecule as claimed in claim 16, wherein said DNA polymerase comprises the amino acid sequence of SEQ ID NO:

3.

19. The isolated nucleic acid molecule as claimed in claim 16, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

2.

20. The isolated nucleic acid molecule as claimed in claim 16, wherein said DNA polymerase has reverse transcriptase activity.

21. The isolated nucleic acid molecule as claimed in claim 16, wherein said DNA polymerase has an enhanced rate of incorporation of ³⁵ S! dATP relative to Thermus aquaticus DNA polymerase.

22. An isolated nucleic acid molecule comprising a gene coding for Thermotoga neapolitana DNA polymerase, wherein said gene coding for Thermotoga neapolitana DNA polymerase has the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4.

23. An isolated nucleic acid molecule comprising a fragment of a gene coding for Thermotoga neapolitana DNA polymerase, wherein said gene coding for Thermotoga neapolitana DNA polymerase has the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4 and said fragment encodes a polypeptide having DNA polymerase activity.

24. The isolated nucleic acid molecule as claimed in claim 23, wherein said polypeptide has reverse transcriptase activity.

25. The isolated nucleic acid molecule as claimed in claim 23, wherein said polypeptide has an enhanced rate of incorporation of ³⁵ S!dATP relative to Thermus aguaticus DNA polymerase.

30. A recombinant host cell transformed with a gene encoding a thermostable Thermotoga neapolitana DNA polymerase, wherein said DNA polymerase is a Pol I-type polymerase.

36. The recombinant host cell as claimed in any one of claims 30 and 31, wherein said host cell is prokaryotic.

37. The recombinant host cell as claimed in claim 36, wherein said host cell is Escherichia coli.

38. The recombinant host cell as claimed in claim 37, wherein said host cell is Escherichia coli DH10B/pUC-Tne as deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, USDA, under Deposit No. NRRL B-21338.

31. A recombinant host cell transformed with a gene encoding a Thermotoga neapolitana DNA polymerase having a molecular weight of about 100 kDa, or fragments thereof having DNA polymerase activity.

32. The recombinant host cell as claimed in claim 31, wherein said recombinant host cell can produce a Thermotoga neapolitana DNA polymerase having the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

33. The recombinant host cell as claimed in claim 31, wherein said recombinant host cell is Escherichia coli DH10B/pUC-Tne.

34. The recombinant host cell as claimed in claim 31, wherein said recombinant host cell comprises a gene for Thermotoga neapolitana DNA polymerase having the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4.

35. The recombinant host cell as claimed in claim 31, wherein said recombinant host cell comprises a fragment of the gene for Thermotoga neapolitana DNA polymerase, wherein said DNA polymerase gene has the restriction map of the Thermotoga neapolitana DNA polymerase gene as shown in FIG. 4.

39. A method of producing a thermostable Thermotoga neapolitana DNA polymerase, said method comprising:

(a) culturing a cell transformed with a gene encoding said thermostable Thermotoga neapolitana DNA polymerase or fragments thereof having DNA polymerase activity;

(b) expressing said gene; and

(c) isolating said DNA polymerase or fragment thereof having DNA polymerase activity from said cell or the cell culture of said cell.

40. The method of producing a thermostable Thermotoga neapolitana DNA polymerase as claimed in claim 39, wherein said cell is a recombinant host cell.

41. The method of producing a thermostable DNA polymerase as claimed in claim 39, wherein said thermostable DNA polymerase has a molecular weight of about 100 kDa.

44. The method of producing a thermostable DNA polymerase as claimed in claim 39, wherein said gene has the restriction map of the Thermotoga neapolitana DNA polymerase as shown in FIG. 4.

45. The method of producing a thermostable DNA polymerase as claimed in claim 39, wherein said host cell is a eukaryotic host cell.

46. The method of producing a thermostable DNA polymerase as claimed in claim 39, wherein said host cell is a prokaryotic host cell.

42. The method of producing a thermostable DNA polymerase as claimed 39, wherein said thermostable DNA polymerase has the amino acid sequence of the Thermotoga neapolitana DNA polymerase produced by Escherichia coli DH10B/pUC-Tne.

43. The method of producing a thermostable DNA polymerase as claimed 40, wherein said recombinant host cell is Escherichia coli DH10B/pUC-Tne.

47. The method of producing a thermostable DNA polymerase as claimed 46, wherein said prokaryotic host cell is Escherichia coli.