T7 DNA polymerase

• US 4,942,130 A
• Filed: 12/14/1987
• Issued: 07/17/1990
• Est. Priority Date: 01/14/1987
• Status: Expired due to Term

• Current Assignee: Harvard University
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Tabor, Stanley, Richardson, Charles C.
• Primary Examiner(s): Mays, Thomas D.
• Application Number:
  US07/132,569
• Time in Patent Office: 946 Days
• Field of Search: 435/6, 435/91, 435/172.1, 435/172.3, 435/184, 435/194, 536/27, 935/14, 935/17
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

The invention relates to methods of DNA sequencing including a method for determining the nucleotide base sequence of a DNA molecule, comprising providing the DNA molecule annealed with a primer molecule able to hybridize to the DNA molecule,   incubating the annealed mixture with a DNA polymerase, except reverse transcriptase, in the presence of four different deoxynucleoside triphosphates and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, the concentration of all four deoxynucleoside triphosphates at the start of said incubating being sufficient to allow DNA synthesis to continue until terminated by said agent. The DNA products of the incubating reaction are separated according to their size, so that at least a part of the nucleotide base sequence of the DNA molecule can be determined. In a furter embodiment the primer may be extended and labelled prior to the incubating reaction.

Claims

1. A purified modified gene encoding a modified bacteriophage T7-type DNA polymerase wherein said polymerase comprises sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity, and wherein said modified gene modified to reduce the activity of naturally-occurring exonuclease activity of the naturally occurring DNA polymerase.

2. A modified bacteriophage T7-type DNA polymerase encoded by the gene of claim 1.

3. The gene of claim 1 encoding a modified bacteriophage T7-type DNA polymerase having a naturally occurring His residue of a T7-type DNA polymerase deleted or replaced with another amino acid to thereby reduce said exonuclease activity.

4. The gene of claim 3, said His residue being His-123 of T7 DNA polymerase.

5. The gene of claim 3 wherein said His residue is deleted.

6. A modified T7-type DNA polymerase encoded by the gene of claim 3, or 5.

7. The gene of claim 1 encoding a modified bacteriophage T7 DNA polymerase having amino acids that correspond to positions 118 and 123 in the naturally occurring T7 DNA polymerase deleted or substituted by other amino acids.

8. The modified gene of claim 1 wherein said naturally occurring gene is gene 5 of bacteriophage T7.

14. The gene of claim 3 encoding a modified bacteriophage T7-type DNA polymerase having a naturally occurring His residue of a T7-type DNA polymerase replaced with a serine or a glutamic acid at position 123.

16. The gene of claim 3 encoding a modified bacteriophage T7-type DNA polymerase having a naturally occurring His residue of a T7-type DNA polymerase replaced with a glutamic acid at position 123 and having a naturally occurring Ser residue of a T7-type DNA polymerase replaced with an alanine at position 122.

17. The gene of claim 1 or 10 encoding a modified bacteriphage T7-type DNA polymerase having a naturally occurring Lys residue at position 118 and a naturally occurring Arg residue at position 119 of a T7-type DNA polymerase each replaced with a glutamic residue.

18. The gene of claim 1 or 10 encoding a modified bacteriphage T7-type DNA polymerase having a naturally occurring Arg residue at position 131, a naturally occurring Lys residue at position 136, a naturally occurring Lys residue at position 140, a naturally occurring Lys residue at position 144, and a naturally occurring Arg residue at position 145, of a T7-type DNA polymerase each replaced with a glutamic residue.

22. A modified T7-type DNA polymerase encoded by the gene of claim 17.

23. A modified T7-type DNA polymerase encoded by the gene of claim 18.

9. A purified modified gene encoding a modified bacteriophage T7-type DNA polymerase wherein said polymerase comprises sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity, and wherein said modified gene results from the modification of a naturally occurring gene modified to eliminate the naturally occurring exonuclease activity of the naturally occurring DNA polymerase.

10. A modified gene encoding a modified bacteriphage T7-type DNA polymerase, wherein said polymerase comprises sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity and wherein said modified gene results from the modification of a naturally occurring gene modified to reduce by at least 30% the naturally occurring exonuclease activity of the naturally occurring DNA polymerase.

11. The modified gene of claim 10 wherein said gene is modified to reduce the level of the naturally occurring exonuclease activity to at least one-fourth that of the naturally occurring polymerase.

12. The modified gene of claim 11 wherein said exonuclease activity is reduced to less than 2% of the naturally occurring activity.

13. A modified bacteriophage T7-type DNA polymerase encoded by the modified gene of claim 8, 9, 10, 11 or 12.

15. The gene of claim 5 or 10 encoding a modified bacteriphage T7-type DNA polymerase lacking the naturally occurring serine residue at position 122 and the naturally occurring histidine residue at position 123.

21. A modified T7-type DNA polymerase encoded by the gene of claim 15.

19. A purified modified T7-DNA polymerase having sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity, wherein said polymerase, compared to native DNA polymerase, lacks one or more amino acids and thereby has a reduced exonuclease activity.

20. A purified modified T7-type DNA polymerase having sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity, wherein said polymerase, compared to native DNA polymerase has one or more amino acids substituted by an amino acid other than that naturally occurring at the site of substitution, and thereby has a reduced exonuclease activity.

24. A modified gene encoding a processive modified DNA polymerase, wherein said polymerase comprises sufficient DNA polymerase activity for use in DNA sequencing when said polymerase is combined with any host factor necessary for said DNA polymerase activity, and wherein said modified gene results from the modification of a naturally occurring gene modified to reduce the activity of naturally occurring exonuclease activity of the naturally occurring DNA polymerase.

25. The gene of claim 24 wherein said gene is modified to completely reduce said exonuclease activity to a level below that of measurable activity.

26. The gene of claim 24 wherein said gene is modified to reduce the level of the naturally occurring exonuclease activity to at least one-fourth that of the naturally occurring polymerase.

27. A processive modified DNA polymerase encoded by the modified gene of claim 24.