US4942130A
Expired / Lapsed
US
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T7 DNA polymerase
Assignee: Harvard University
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Filing: 1987-12-14
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Granted: 1990-07-17
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Expires: 2007-12-14
Abstract
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.
Inventors
- 👤 Stanley Tabor
- 👤 Charles C. Richardson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 13:02
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/52; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q1/6844; C12Q1/686; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
59 words
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.
Claim 2
Dependent Claim
59 words
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.
Claim 3
Dependent Claim
13 words
A modified bacteriophage T7-type DNA polymerase encoded by the gene of claim 1.
Claim 4
Dependent Claim
36 words
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.
Claim 5
Dependent Claim
14 words
The gene of claim 3, said His residue being His-123 of T7 DNA polymerase.
Claim 6
Dependent Claim
11 words
The gene of claim 3 wherein said His residue is deleted.
Claim 7
Dependent Claim
14 words
A modified T7-type DNA polymerase encoded by the gene of claim 3, or 5.
Claim 8
Dependent Claim
36 words
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.
Claim 9
Dependent Claim
17 words
The modified gene of claim 1 wherein said naturally occurring gene is gene 5 of bacteriophage T7.
Claim 10
Dependent Claim
67 words
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.
Claim 11
Dependent Claim
67 words
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.
Claim 12
Dependent Claim
70 words
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.
Claim 13
Dependent Claim
70 words
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.
Claim 14
Dependent Claim
31 words
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.
Claim 15
Dependent Claim
21 words
The modified gene of claim 11 wherein said exonuclease activity is reduced to less than 2% of the naturally occurring activity.
Claim 16
Dependent Claim
19 words
A modified bacteriophage T7-type DNA polymerase encoded by the modified gene of claim 8, 9, 10, 11 or 12.
Claim 17
Dependent Claim
34 words
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.
Claim 18
Dependent Claim
32 words
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.
Claim 19
Dependent Claim
50 words
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.
Claim 20
Dependent Claim
43 words
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.
Claim 21
Dependent Claim
67 words
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.
Claim 22
Dependent Claim
51 words
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.
Claim 23
Dependent Claim
51 words
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.
Claim 24
Dependent Claim
67 words
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.
Claim 25
Dependent Claim
67 words
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.
Claim 26
Dependent Claim
12 words
A modified T7-type DNA polymerase encoded by the gene of claim 15.
Claim 27
Dependent Claim
12 words
A modified T7-type DNA polymerase encoded by the gene of claim 17.
Claim 28
Dependent Claim
12 words
A modified T7-type DNA polymerase encoded by the gene of claim 18.
Claim 29
Dependent Claim
67 words
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.
Claim 30
Dependent Claim
67 words
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.
Claim 31
Dependent Claim
24 words
The gene of claim 24 wherein said gene is modified to completely reduce said exonuclease activity to a level below that of measurable activity.
Claim 32
Dependent Claim
30 words
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.
Claim 33
Dependent Claim
13 words
A processive modified DNA polymerase encoded by the modified gene of claim 24.
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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) | 1993-07-16 | 1994-01-15 | 1994-07-17 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-07-16 | 1998-01-15 | 1998-07-17 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-07-16 | 2002-01-15 | 2002-07-17 | $7700.00 | Window Closed / Lapsed |