Cloned DNA polymerases from Thermotoga and mutants thereof

• US 6,506,560 B1
• Filed: 01/28/1999
• Issued: 01/14/2003
• 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
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Hutson, Richard G.
• Application Number:
  US09/238,471
• Time in Patent Office: 1,447 Days
• Field of Search: 435/6, 435/15, 435/194, 435/320.1, 435/252.3, 435/254.11, 435/325, 435/91.1, 435/91.2, 435/91.5, 435/91.51, 53/231, 53/232
• 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. An isolated Thermotoga neopolitana (Tne) DNA polymerase, wherein said Tne DNA polymerase is a Pol I-type 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.

5. A vector comprising a gene encoding the DNA polymerase of claim 1.

6. The vector of claim 5, wherein said gene is operably linked to a promoter.

7. The vector of claim 6, 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.

8. A host cell comprising the vector of claim 5.

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

(a) culturing the host cell of claim 8;

(b) expressing said gene; and

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

10. The method of claim 9, wherein said host cell is E. coli.

16. 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

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

17. 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 of claim 1, dATP, dCTP, dGTP and dTTP, and an oligo nucleotide or primer which is complementary to the first strand cDNA;

whereby dsDNA is obtained.

4. An isolated Thermotoga neopolitana (Tne) DNA polymerase having the amino acid sequence of SEQ ID NO:

3.

11. 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 dedioxy ribonucleoside triphosphates and the DNA polymerase of claim 4, under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.

12. The method of claim 11, 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.

13. 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 claim 4, under conditions such that a third strand complementary to said first strand and a fourth strand 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.

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

(a) a first container means comprising the DNA polymerase of claim 4;

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

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

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

(a) a first container means comprising the DNA polymerase of claim 4; and

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