T7 DNA polymerase

• US 4,946,786 A
• Filed: 03/01/1990
• Issued: 08/07/1990
• 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:
  US07/487,746
• Time in Patent Office: 159 Days
• Field of Search: 435/172.3, 435/194, 435/252.33, 435/320, 935/14, 935/29
• 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

Abstract

1Method for production of a composition consisting essentially of a T7-type DNA polymerase and thioredoxin. The method includes culturing a cell containing plasmid DNA encoding a T7-type DNA polymerase to express the T7-type DNA polymerase from the plasmid DNA, and purifying the T7-type DNA polymerase expressed from the cell to reduce the exonuclease activity associated with the T7-type DNA polymerase compared to the level of exonuclease activity associated with a corresponding naturally-occurring T7-type DNA polymerase.

Claims

1. A method for production of a composition consisting essentially of a T7-type DNA polymerase and thioredoxin, comprising the steps of:

culturing a cell comprising plasmid DNA encoding a T7-type DNA polymerase to express said T7-type DNA polymerase from said plasmid DNA, and

purifying said T7-type DNA polymerase expressed from said cell to reduce the exonuclease activity associated with said T7-type DNA polymerase to a level below that of exonuclease activity associated with a corresponding naturally-occurring T7-type DNA polymerase.

2. The method of claim 1 further comprising providing thioredoxin, and contacting said thioredoxin with said T7-type DNA polymerase.

3. The method of claim 1 wherein said purifying comprises reducing the exonuclease activity associated with said T7-type DNA polymerase to less than 500 units of activity.

4. The method of claim 2 wherein said thioredoxin is provided by culturing a cell comprising plasmid DNA encoding said thioredoxin to express said thioredoxin from said cloned DNA.

5. The method of claim 4 wherein said cloned DNA is provided in said cell within a plasmid.

6. The method of claim 1 wherein said purifying comprises contacting said DNA polymerase with iron ions at a concentration sufficient to reduce the activity of said exonuclease.

7. The method of claim 1 wherein said purifying comprises contacting said DNA polymerase with an oxygen radical at a concentration sufficient to reduce the activity of said exonuclease.

8. A composition consisting essentially of:

a T7-type /DNA polymerase produced by the method of claim 1 or 2.

9. The composition of claim 8 further consisting essentially of thioredoxin.

10. The composition of claim 8, wherein said polymerase has less than 500 units of exonuclease activity per milligram of said polymerase.

11. The composition of claim 8 wherein said plasmid DNA comprises gene 5 of bacteriophage T7.

12. The composition of claim 8 wherein said exonuclease activity is reduced to less than one percent of the naturally occurring activity of the naturally occurring DNA polymerase.

13. The composition of claim 8 wherein said T7-type DNA polymerase is selected from the group consisting of the DNA polymerases of phage T7, T3, Φ

I, Φ

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

14. The composition of claim 8 wherein said T7-type DNA polymerase is T7 DNA polymerase.

15. The composition of claim 14 wherein said T7 DNA polymerase has less than one percent of the exonuclease activity of the naturally associated level of exonuclease activity of the naturally occurring DNA polymerase.

16. A method for production of a composition comprising purified T7-type DNA polymerase at a concentration suitable for use in DNA sequencing, comprising the steps of:

providing a T7-type DNA polymerase and thioredoxin together having sufficient DNA polymerase activity for use in DNA sequencing, wherein said T7-type DNA polymerase is expressed from cloned DNA, and

treating said T7-type DNA polymerase to reduce the exonuclease activity associated with said T7-type DNA polymerase to a level below that of exonuclease activity associated with a corresponding naturally occurring T7-type DNA polymerase.

18. A composition consisting essentially of a T7 DNA polymerase produced by the method of claim 16 or 17.

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17. A method for production of a composition comprising purified T7-type DNA polymerase at a concentration suitable for use in DNA sequencing, comprising the steps of:

providing a T7-type DNA polymerase having sufficient DNA polymerase activity for use in DNA sequencing, wherein said T7-type DNA polymerase is expressed from cloned DNA, and

treating said T7-type DNA polymerase to reduce the exonuclease activity associated with said T7-type DNA polymerase to a level below that of exonuclease activity associated with a corresponding naturally occurring T7-type DNA polymerase.