Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension

• US 5,436,149 A
• Filed: 02/19/1993
• Issued: 07/25/1995
• Est. Priority Date: 02/19/1993
• Status: Expired due to Term

• Current Assignee: Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Wayne M. Barnes
• Inventors: Barnes, Wayne M.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/021,623
• Time in Patent Office: 886 Days
• Field of Search: 435/91, 435/194, 435/172..3, 435/252.1, 435/91.1, 435/91.2, 435/91.4, 435/91.5, 435/193, 935/16
• 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 1/686 Polymerase chain reaction [PCR]

Abstract

A DNA polymerase having an amino acid sequence comprising substantially the same amino acid sequence as that of Thermus aquaticus or Thermus flavus DNA polymerase, excluding the N-terminal 280 amino acid residues of Thermus aquaticus DNA polymerase or the N-terminal 279 amino acid residues of Thermus flavus DNA polymerase, recombinant DNA sequences encoding said DNA polymerases, vectors comprising said DNA sequences, and host cells containing such vectors. A formulation of thermostable DNA polymerases comprising a majority component comprised of at least one thermostable DNA polymerase of the type described above, wherein the DNA polymerase lacks 3'"'"'-exonuclease activity, and a minority component comprised of at least one thermostable DNA polymerase exhibiting 3'"'"'-exonuclease activity, and an improved method for amplifying nucleic acid sequences by polymerase chain reaction wherein the above formulation is mixed and used to catalyze primer extension, are also provided.

Claims

1. A DNA polymerase comprising substantially the same amino acid sequence as that of Thermus aquaticus DNA polymerase, excluding the N-terminal 280 amino acid residues of Thermus aquaticus DNA polymerase.

2. A DNA polymerase as set forth in claim 1, consisting of the amino acid sequence of SEQ ID NO:

6.

3. A DNA polymerase as set forth in claim 1 that is encoded by plasmid pWB254b.

4. A DNA polymerase as set forth in claim 1 wherein the DNA polymerase has been purified to be substantially free of other proteins.

7. A formulation of thermostable DNA polymerases as set forth in claim 6 wherein the at least one thermostable DNA polymerase lacking 3'"'"'-exonuclease activity is the DNA polymerase set forth in claim 1.

9. A formulation of thermostable DNA polymerases as set forth in claim 7 wherein the DNA polymerases are present in a ratio of from about 10 units to about 2000 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

10. A formulation of thermostable DNA polymerases as set forth in claim 9 wherein the DNA polymerases are present in a ratio of about 640 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

5. A DNA polymerase comprising substantially the same amino acid sequence as that of Thermus flavus DNA polymerase, excluding the N-terminal 279 amino acid residues of Thermus flavus DNA polymerase.

14. A formulation of thermostable DNA polymerases as set forth in claim 6 wherein the at least one thermostable DNA polymerase lacking 3'"'"'-exonuclease activity is the DNA polymerase set forth in claim 5.

15. A formulation of thermostable DNA polymerases as set forth in claim 14 wherein the DNA polymerases are present in a ratio of from about 10 units to about 2000 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

16. A formulation of thermostable DNA polymerases as set forth in claim 15 wherein the DNA polymerases are present in a ratio of about 640 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

6. A formulation of thermostable DNA polymerases comprising at least one thermostable DNA polymerase lacking 3'"'"'-exonuclease activity and at least one thermostable DNA polymerase exhibiting 3'"'"'-exonuclease activity, wherein the DNA polymerases are present in a ratio of from about 4 units to about 2000 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

8. A formulation of thermostable DNA polymerases as set forth in claim 6 wherein the at least one thermostable DNA polymerase exhibiting 3'"'"'-exonuclease activity is selected from the group consisting of Pfu polymerase from Pyroccus furiosus, the Vent DNA polymerase from Thermococcus litoralis, a variant of the Pfu DNA polymerase wherein the DNA polymerase activity of said Pfu DNA polymerase has been diminished or inactivated, or a variant of the Vent DNA polymerase wherein the DNA polymerase activity of said Vent DNA polymerase has been diminished or inactivated.

11. A formulation of DNA polymerases as set forth in claim 6 wherein the DNA polymerases are present in a ratio of from about 10 units to about 2000 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

12. A formulation of DNA polymerases as set forth in claim 6 wherein the DNA polymerases are present in a ratio of from about 80 units to about 1000 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.

13. A formulation of DNA polymerases as set forth in claim 6 wherein the DNA polymerases are present in a ratio of from about 640 units of the DNA polymerase lacking 3'"'"'-exonuclease activity to 1 unit of the DNA polymerase exhibiting 3'"'"'-exonuclease activity.