Thermostable DNA polymerases

• US 5,885,813 A
• Filed: 05/14/1996
• Issued: 03/23/1999
• Est. Priority Date: 05/31/1995
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Amersham Life Science, Inc. (GE Healthcare Technologies, Inc.)
• Inventors: Fuller, Carl W., Davis, Maria, Moffett, R. Bruce, Cunniff, John J.
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/648,657
• Time in Patent Office: 1,043 Days
• Field of Search: 435/183, 435/194, 435/810, 435/91.1, 435/91.2
• US Class Current: 435/183
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

An enzymatically active DNA polymerase having between 540 and 582 amino acids having a tyrosine at a position equivalent to position 667 of Taq DNA polymerase, wherein said polymerase lacks 5'"'"' to 3'"'"' exonuclease activity, and wherein said polymerase has at least 95% homology in its amino acid sequence to the DNA polymerase of Thermus aquaticus, Thermus flavus or Thermus thermophilus, and wherein said polymerase forms a single polypeptide band on an SDS PAGE.

Claims

1. An enzymatically active DNA polymerase comprising 561 amino acids having a tyrosine residue at an amino acid position corresponding to Taq DNA polymerase residue 667 in its dNMP binding site, wherein said polymerase lacks 5'"'"' to 3'"'"' exonuclease activity as a result of an N-terminal deletion, and wherein said polymerase is substantially identical to the DNA polymerase of Thermus aquaticus, Thermus flavus or Thermus thermophilus, and wherein said polymerase forms a single polypeptide band on an SDS polyacrylamide gel.

4. The polymerase of claim 1, wherein the polymerase is FY4 (SEQ ID NO:

14).

5. The polymerase of claim 1 or 2 wherein the amino acid sequence of said polymerase includes less than 3 conservative amino acid changes compared to the equivalent region of one said DNA polymerase of said named Thermus species.

6. The polymerase of claim 1 or 2 wherein the amino acid sequence of said polymerase includes less than 3 additional amino acids at its N-terminus compared to the equivalent region of one said DNA polymerase of said named Thermus species.

7. The polymerase of claim 1 wherein the polymerase is FY2 (SEQ ID NO:

1).

8. Purified nucleic acid encoding the DNA polymerase of any of claims 1, 7, 2, 3, or 4.

9. Method for sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with a DNA polymerase of any of claims 1, 7, 2, 3, or 4 in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments.

10. Kit for sequencing DNA comprising a DNA polymerase of any of claims 1, 7, 2, 3, or 4 and a pyrophosphatase.

11. The kit of claim 10 wherein said pyrophosphatase is thermostable.

12. Apparatus for DNA sequencing having a reactor comprising a DNA polymerase of any of claims 1, 7, 2, 3, or 4 and a band separator.

13. Purified nucleic acid encoding the DNA polymerase of claim 5.

14. Method for sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with a DNA polymerase of claim 5 in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments.

15. Kit for sequencing DNA comprising a DNA polymerase of claim 5 and a pyrophosphatase.

16. The kit of claim 15 wherein said pyrophosphatase is thermostable.

17. Apparatus for DNA sequencing having a reactor comprising a DNA polymerase of claim 5 and a band separator.

18. Purified nucleic acid encoding the DNA polymerase of claim 6.

19. Method for sequencing DNA comprising the step of generating chain terminated fragments from the DNA template to be sequenced with a DNA polymerase of claim 6 in the presence of at least one chain terminating agent and one or more nucleotide triphosphates, and determining the sequence of said DNA from the sizes of said fragments.

20. Kit for sequencing DNA comprising a DNA polymerase of claim 6 and a pyrophosphatase.

21. The kit of claim 20 wherein said pyrophosphatase is thermostable.

22. Apparatus for DNA sequencing having a reactor comprising a DNA polymerase of claim 6 and a band separator.

2. An enzymatically active DNA polymerase comprising 562 amino acids having a tyrosine residue at an amino acid position corresponding to Taq DNA polymerase residue 667 in its dNMP binding site, wherein said polymerase lacks 5'"'"' to 3'"'"' exonuclease activity as a result of an N-terminal deletion, and wherein said polymerase is substantially identical to the DNA polymerase of Thermus aquaticus, Thermus flavus or Thermus thermophilus, and wherein said polymerase forms a single polypeptide band on an SDS polyacrylamide gel.

3. The polymerase of claim 2, wherein the polymerase is FY3 (SEQ ID NO:

2).