T7 DNA polymerase

• US 4,921,794 A
• Filed: 12/05/1988
• Issued: 05/01/1990
• Est. Priority Date: 01/14/1987
• Status: Expired due to Term

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Tabor, Stanley, Richardson, Charles C.
• Primary Examiner(s): Mays, Thomas D.
• Application Number:
  US07/279,990
• Time in Patent Office: 512 Days
• Field of Search: 435/91, 435/6, 435/21, 435/172.3, 935/14, 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

This invention relates to T7-type DNA polymerases and methods for amplification of DNA, for example by polymerase chain reaction.

Claims

1. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a T7- type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence with their 3'"'"' ends directed towards each other after annealing, and with the DNA sequence to be amplified located between the two annealed primers.

2. The method of claim 1 wherein said method further comprises, after said incubating step, denaturing the resulting DNA, annealing said first and second primers to said resulting DNA and incubating the annealed mixture with said polymerase.

3. The method of claim 2 wherein said steps of denaturing, then annealing and then incubating are repeated from 10 to 40 times.

4. The method of claim 1 wherein said exonuclease activity is less than 1 unit per mg of polymerase.

5. The method of claim 1, said T7- type DNA polymerase being selected from the group consisting of the DNA polymerases of phage T7, T3, Φ

, Φ

II, H, W31, gh-1, Y, A1122 and Sp6.

6. The method of claim 1 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.

7. The method of claim 1 wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.

8. The method of claim 7 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.

9. The method of claim 7 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.

10. The method of claim 1 wherein said polymerase has no detectable exonuclease activity.

11. The method of claim 1 wherein said first and second primers include 10 bases or more.

12. The method of claim 1 wherein said first and second primers include 4 bases or more.

13. The method of claim 1 wherein said first and second primers comprise 4 to 20 bases.

14. The method of claim 1 wherein said polymerase is non-discriminating for nucleotide analogs and

said first and second primers are single-stranded RNA or DNA comprising 4-10 bases.

15. The method of claim 1 wherein said first and second primers are single stranded DNA or RNA.

16. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive T7-type DNA polymerase, having less than 50% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

17. The method of claim 16 wherein said polymerase has less than 1% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

18. The method of claim 16 wherein said T7- type DNA polymerase is T7 DNA polymerase.

19. The method of claim 18 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

20. A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive T7- type DNA polymerase, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined with said polymerase.

21. The method of claim 20 wherein said T7- type DNA polymerase is T7 DNA polymerase.

22. The method of claim 20 wherein said processive DNA polymerase has less than one percent of the exonuclease activity of the naturally occurring level of exonuclease activity of said polymerase.

23. The method of claim 22 wherein said polymerase has no detectable exonuclease activity.

24. The method of claim 22 or 23 wherein said polymerase is T7 DNA polymerase.