US4946786A
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US
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T7 DNA polymerase
Assignee: Harvard University
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Filing: 1990-03-01
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Granted: 1990-08-07
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Expires: 2010-03-01
Abstract
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.
Inventors
- 👤 Stanley Tabor
- 👤 Charles C. Richardson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 12:22
CPC Classifications
C; C12; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
78 words
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.
Claim 2
Independent Claim
78 words
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.
Claim 3
Dependent Claim
18 words
The method of claim 1 further comprising providing thioredoxin, and contacting said thioredoxin with said T7-type DNA polymerase.
Claim 4
Dependent Claim
26 words
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.
Claim 5
Dependent Claim
28 words
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.
Claim 6
Dependent Claim
17 words
The method of claim 4 wherein said cloned DNA is provided in said cell within a plasmid.
Claim 7
Dependent Claim
27 words
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.
Claim 8
Dependent Claim
28 words
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.
Claim 9
Dependent Claim
18 words
A composition consisting essentially of: a T7-type /DNA polymerase produced by the method of claim 1 or 2.
Claim 10
Dependent Claim
10 words
The composition of claim 8 further consisting essentially of thioredoxin.
Claim 11
Dependent Claim
21 words
The composition of claim 8, wherein said polymerase has less than 500 units of exonuclease activity per milligram of said polymerase.
Claim 12
Dependent Claim
15 words
The composition of claim 8 wherein said plasmid DNA comprises gene 5 of bacteriophage T7.
Claim 13
Dependent Claim
27 words
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.
Claim 14
Dependent Claim
33 words
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, ΦI, ΦII, H, W31, gh-1, Y, A1122 and Sp6.
Claim 15
Dependent Claim
14 words
The composition of claim 8 wherein said T7-type DNA polymerase is T7 DNA polymerase.
Claim 16
Dependent Claim
33 words
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.
Claim 17
Independent Claim
87 words
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.
Claim 18
Independent Claim
87 words
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.
Claim 19
Independent Claim
84 words
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.
Claim 20
Independent Claim
84 words
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.
Claim 21
Dependent Claim
38 words
A composition consisting essentially of a T7 DNA polymerase produced by the method of claim 16 or 17. If there is any fee in connection with this Preliminary Amendment, please charge Deposit Account 06-1050 for the appropriate amount.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-08-06 | 1994-02-05 | 1994-08-07 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-08-06 | 1998-02-05 | 1998-08-07 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-08-06 | 2002-02-05 | 2002-08-07 | $7700.00 | Window Closed / Lapsed |