Cloned DNA polymerases from Thermotoga and mutants thereof

• US 20030162201A1
• Filed: 11/01/2002
• Published: 08/28/2003
• Est. Priority Date: 09/30/1994
• Status: Abandoned Application

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Chatterjee, Deb K., Hughes, A. John Jr.
• Application Number:
  US10/285,696
• Publication Number:
  US 20030162201A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 substantially pure Thermotoga neapolitana (Tne) DNA polymerase.

2. The DNA polymerase of claim 1, which is isolated from Thermotoga neapolitana.

3. The DNA polymerase of claim 2, which is isolated from Thermotoga neapolitana DSM 5068 and said DNA polymerase has a molecular weight of about 100 kilodaltons.

4. The DNA polymerase of claim 1 having the amino acid sequence of SEQ ID NO:

3.

17. A vector comprising a gene encoding the DNA polymerase of any one of claims 1 or 5.

18. The vector of claim 17, wherein said gene is operably linked to a promoter.

19. The vector of claim 18, wherein said promoter is selected from the group consisting of a λ

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

20. A host cell comprising the vector of claim 17.

21. A method of producing a DNA polymerase, said method comprising:

(a) culturing the host cell of claim 20;

(b) expressing said gene; and

(c) isolating said DNA polymerase from said host cell.

22. The method of claim 21, wherein said host cell is E. coli.

23. 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 step (a) in the presence of one or more deoxy- or dideoxyribonucleoside triphosphates and the DNA polymerase of any one of claims 1 or 5, under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.

24. The method of claim 21, wherein said deoxy- or dideoxyribonucleoside 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.

25. The method of claim 23, wherein one or more of said deoxy- or dideoxyribonucleoside triphosphates are detectably labeled.

(a) A method of sequencing a DNA molecule, comprising:

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

(b) contacting said DNA molecule of step (a) with deoxyribonucleoside triphosphates, the DNA polymerase of any one of claims 1 or 5, 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 5′

termini; 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.

30. A method for 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 the DNA polymerase of any one of claims 1 or 5, 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.

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

(a) a first container means comprising the DNA polymerase of any one of claims 1 or 5;

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

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

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

(a) a first container means comprising the DNA polymerase of any one of claims 1 or 5; and

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

38. A method of preparing cDNA from mRNA, comprising

(a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid, and

(b) contacting said hybrid formed in step (a) with the Tne DNA polymerase or mutant of claim 1 or 5 and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid is obtained.

39. A method of preparing dsDNA from mRNA, comprising

(a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid, and

(b) contacting said hybrid formed in step (a) with the Tne DNA polymerase or mutant of claim 1 or 5, dATP, dCTP, dGTP and dTTP, and an oligonucleotide or primer which is complementary to the first strand cDNA;

whereby dsDNA is obtained.

5. A Tne DNA polymerase mutant which is modified at least one way selected from the group consisting of

(a) to reduce or eliminate the 3′

→

5′

exonuclease activity of the polymerase;

(b) to reduce or eliminate the 5′

→

3′

exonuclease activity of the polymerase; and

(c) to reduce or eliminate discriminatory behavior against a dideoxynucleotide.

6. The DNA polymerase mutant of claim 5, which is modified at least two ways.

7. The DNA polymerase mutant of claim 5, which is modified three ways.

8. The Tne DNA polymerase mutant of claim 5 which comprises a mutation in the O-helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides.

9. The DNA polymerase of claim 8, wherein said O-helix is defined as RXXXKXXXFXXXYX, wherein X is any amino acid.

11. The Tne DNA polymerase of claim 5, wherein said DNA polymerase is a Tne DNA polymerase having substantially reduced 3′

→

5′

exonuclease activity.

12. The mutant Tne DNA polymerase as claimed in claim 11, wherein said mutant is a Asp³²³→

Ala³²³ substitution.

13. The mutant Tne DNA polymerase as claimed in claim 5, wherein said mutant polymerase comprises both a Phe⁷³⁰→

Tyr⁷³⁰ substitution and a Asp³²³→

Ala³²³ substitution.

14. The mutant DNA polymerase mutant of claim 5, wherein said DNA polymerase is a Tne DNA polymerase having substantially reduced 5′

→

3′

exonuclease activity.

15. The mutant Tne DNA polymerase as claimed in claim 14, wherein said mutant polymerase has a deletion mutation in the N-terminal 5′

→

3′

exonuclease domain.

16. The mutant Tne DNA polymerase as claimed in claim 15, wherein said mutant polymerase is devoid of the 219 N-terminal amino acids.

10. The Tne DNA polymerase as claimed in claim 10, wherein said mutation is a Phe⁷³⁰→

Tyr⁷³⁰ substitution.

26. The method of claim 26, 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.

27. The method of claim 26, wherein said terminator nucleotide is ddTTP, ddATP, ddGTP, ddITP or ddCTP.

28. The method of claim 26, wherein one or more of said deoxyribonucleoside triphosphates is detectably labeled.

29. The method of claim 26, wherein one or more of said terminator nucleotides is detectably labeled.

31. The method of claim 31, 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.

34. A mutant Tne DNA polymerase having substantially reduced or eliminated 5′

-3′

exonuclease activity, wherein at least one of the amino acids corresponding to Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, Asp¹³⁹, Gly¹⁰², Gly¹⁸⁷, or Gly¹⁹⁵ has been mutated.

35. A vector coding for the mutant DNA polymerase of claim 35.

36. A host cell comprising the vector of claim 36.

37. A method of producing a mutant Tne DNA polymerase having substantially reduced or eliminated 5′

-3′

exonuclease activity, wherein at least one of the amino acids corresponding to Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, Asp¹³⁹, Gly¹⁰², Gly¹⁸⁷, or Gly¹⁹⁵ has been mutated, comprising

(a) culturing the host cell of claim 37;

(b) expressing the mutant DNA polymerase;

amd

(c) isolating said mutant DNA polymerase.