Polymerase compositions and uses thereof

• US 5,556,772 A
• Filed: 02/16/1994
• Issued: 09/17/1996
• Est. Priority Date: 12/08/1993
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc., Takara Bio Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., Mullinax, Rebecca L.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/197,791
• Time in Patent Office: 944 Days
• Field of Search: 435/91.2, 435/6, 935/77, 935/78
• US Class Current: 435/91.2
• 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

The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3'"'"'-5'"'"' exonuclease activity (b) a DNA polymerase with less 3'"'"'-5'"'"' exonuclease activity than the enzyme with substantial 3'"'"'-5'"'"' exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3'"'"'-5'"'"' exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3'"'"'-5'"'"' exonuclease activity and a DNA polymerase with less 3'"'"'-5'"'"' exonuclease activity than the enzymes possessing substantial 3'"'"'-5'"'"' exonuclease activity, preferably a DNA polymerase that substantially lacks 3'"'"'-5'"'"' exonuclease activity. Another aspect of the invention involves the use the subject method of polynucleotide synthesis to carry out the synthesis step in a polymerase chain reaction experiment. Yet another aspect of the invention is to provide kits for the synthesis of polynucleotides, wherein the kits comprise an enzyme that possesses substantial 3'"'"'-5'"'"' exonuclease activity and a DNA polymerase with less 3'"'"'-5'"'"' exonuclease activity than the enzyme possessing substantial 3'"'"'-5'"'"' exonuclease activity.

Claims

1. A kit for the synthesis of a polynucleotide, said kit comprising:

(a) a first DNA polymerase, wherein said first polymerase possesses 3'"'"'-5'"'"' exonuclease activity selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase, and

(b) a second DNA polymerase, wherein said second polymerase lacks 3'"'"'-5'"'"' exonuclease activity selected from the group consisting of Thermus aquaticus DNA polymerase, (exo-) Thermococcus litoralis DNA polymerase, (exo-) Pyrococcus furiosus DNA polymerase, and (exo-) Pyrococcus GB-D DNA polymerase.

2. A kit according to claim 1, wherein said first and second DNA polymerases are thermostable.

8. A kit according to claim 2, wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.

9. A kit according to claim 2, wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.

10. A kit according to claim 8, wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.

11. A kit according to claim 1, said kit further comprising DNA primers.

12. A kit according to claim 2, said kit further comprising DNA primers.

13. A kit according to claim 10, said kit further comprising DNA primers.

3. A method of amplifying a polynucleotide sequence, said method comprising:

the steps of mixing a composition, with a synthesis primer, and a synthesis template, said composition comprising

(a) a first DNA polymerase possessing 3'"'"'-5'"'"' exonuclease activity selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase, and

(b) a second DNA polymerase, wherein said polymerase lacks 3'"'"'-5'"'"' exonuclease activity selected from the group consisting of Thermus aquaticus DNA polymerase, (exo-) Thermococcus litoralis DNA polymerase, (exo-) Pyrococcus furiosus DNA polymerase, and (exo-) Pyrococcus GB-D DNA polymerase.

4. A method according to claim 3 wherein said first and second DNA polymerases are thermostable.

5. A method according to claim 3, wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.

6. A method according to claim 4, wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.

7. A method according to claim 5, wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.