Cloned DNA polymerases from Thermotoga neapolitana

• US 6,444,424 B1
• Filed: 01/26/1999
• Issued: 09/03/2002
• Est. Priority Date: 09/30/1994
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K., Hughes, A. John Jr.
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): HUTSON, RICHARD G
• Application Number:
  US09/236,615
• Time in Patent Office: 1,316 Days
• Field of Search: 435/194, 435/6, 435/91.1, 435/91.2, 435/91.5, 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 1/6844 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 method of synthesizing a double-stranded DNA molecule comprising:

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

(b) incubating said DNA molecule of (a) in the presence of one or more deoxyribonucleoside triphosphates and Tne DNA polymerase, wherein said Tne DNA polymerase is a Pol I type DNA polymerase, under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.

2. The method of claim 1, wherein said DNA polymerase is isolated from a Thermotoga neapolitana culture.

3. The method of claim 1, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

4. The method of claim 1, wherein said deoxyribonucleoside triphosphates are selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP, ddCTP, ddGTP, ddlTP, ddTTP, [α

S]dATP, [α

S]dTTP, [α

S]dGTP, and [α

S]dCTP.

5. The method of claim 4, wherein one or more of said deoxyribonucleoside triphosphates are detectably labeled.

6. The method of claim 5, wherein said detectable label is selected from the group consisting of a radioactive isotope, a fluorescent label, a chemiluminescent label, a bioluminescent label, and an enzyme label.

23. The method of claim 2, wherein said DNA polymerase is isolated from Thermotoga neapolitana DMS 5068.

24. The method of claim 3, wherein said host is a eukaryotic host.

25. The method of claim 3, wherein said host is a prokaryotic host.

29. The method of claim 1, wherein said Tne DNA polymerase has reduced or eliminated 3′

-5′

exonuclease activity.

7. A method of sequencing a DNA molecule, comprising

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

(b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, Tne DNA polymerase, wherein said Tne DNA polymerase is a Pol I type DNA polymerase, and a terminator nucleotide;

(c) incubating the mixture of step (b) under conditions sufficient to synthesize a random population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 3′

termnini; and

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

8. The method of claim 7, wherein said terminator nucleotide is selected from the group consisting of ddTTP, ddATP, ddGTP and ddCTP.

9. The method of claim 7, wherein said DNA polymerase is isolated from a Thermotoga neapolitana culture.

10. The method of claim 7, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

11. The method of claim 7, wherein one or more of said deoxyribonucleoside triphosphates is detectably labeled.

12. The method of claim 11, wherein said labeled deoxyribonucleoside triphosphate is [α

³⁵S]dATP.

26. The method of claim 9, wherein said DNA polymerase is isolated from Thermotoga neapolitana DMS 5068.

30. The method of claim 7, wherein said Tne DNA polymerase has reduced or eliminated 3′

-5′

exonuclease activity.

13. 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 Tne DNA polymerase, wherein said Tne DNA polymerase is a Pol I type 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 with heat; and

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

14. The method of claim 13, wherein said DNA polymerase is isolated from a Thermotoga neapolitana culture.

15. The method of claim 14, wherein said DNA polymerase is isolated from Thermotoga neapolitana DMS 5068.

16. The method of claim 13, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

31. The method of claim 13, wherein said Tne DNA polymerase has reduced or eliminated 3′

-5′

exonuclease activity.

17. A kit for sequencing a DNA molecule, comprising:

(a) a first container means comprising a Tne DNA polymerase, wherein said Tne DNA polymerase is a Pol I type DNA polymerase

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

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

18. The kit of claim 17, wherein said DNA polymerase is isolated from a Thermotoga neapolitana culture.

19. The kit of claim 17, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

27. The kit of claim 18, wherein said DNA polymerase is isolated from Thermotoga neapolitana DMS 5068.

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

(a) a first container means comprising a Tne DNA polymerase, wherein said Tne DNA polymerase is a Pol I type polymerase, and

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

21. The kit of claim 20, wherein said DNA polymerase is isolated from a Thermotoga neapolitana culture.

22. The kit of claim 20, wherein said DNA polymerase is isolated from a recombinant host expressing a gene encoding said DNA polymerase.

28. The kit of claim 21, wherein said DNA polymerase is isolated from Thermotoga neapolitana DMS 5068.