US4795699A
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T7 DNA polymerase
Assignee: Harvard University
•
Filing: 1987-01-14
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Granted: 1989-01-03
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Expires: 2007-01-14
Abstract
Abstract This invention relates to T7-type DNA polymerases and method for using them.
Inventors
- 👤 Stanley Tabor
- 👤 Charles C. Richardson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 13:26
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; C12Q1/6883; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12P; C12P19/00; C12P19/26; C12P19/28; C12P19/30; C12P19/34; C12Q1/6844; C12Q1/6858; C12Q1/686; C12Q1/6869; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/81
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
156 words
A method for determining the nucleotide base sequence of a DNA molecule, comprising: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule; incubating separate portions of the annealed mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, a processive T7-type DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerse having less than 500 units of exonuclease activity per mg of said polymerase, and one of four DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each said agent terminates DNA synthesis at a different nucleotide base, and separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.
Claim 2
Independent Claim
156 words
A method for determining the nucleotide base sequence of a DNA molecule, comprising: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule; incubating separate portions of the annealed mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, a processive T7-type DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerse having less than 500 units of exonuclease activity per mg of said polymerase, and one of four DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each said agent terminates DNA synthesis at a different nucleotide base, and separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.
Claim 3
Dependent Claim
30 words
The method of claim 1, wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequence reaction.
Claim 4
Dependent Claim
21 words
The method of claim 1 wherein said polymerase remains bound to said DNA molecule for at least 1,000 bases before dissociating.
Claim 5
Dependent Claim
18 words
The method of claim 1 wherein said polymerase is that polymerase in cells infected with a T7-type phage.
Claim 6
Dependent Claim
21 words
The method of claim 4 wherein said T7-type phage is T7, T3, ΦI, ΦII, H, W31, gh-1, Y, Al122 or Sp6.
Claim 7
Dependent Claim
14 words
The method of claim 1 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.
Claim 8
Dependent Claim
24 words
The method of claim 1 wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.
Claim 9
Dependent Claim
20 words
The method of claim 7 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.
Claim 10
Dependent Claim
20 words
The method of claim 7 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.
Claim 11
Dependent Claim
13 words
The method of claim 1 wherein said polymerase has no detectable exonuclease activity.
Claim 12
Dependent Claim
19 words
The method of claim 1 wherein said polymerase is able to utilize primers of 10 base pairs or more.
Claim 13
Dependent Claim
19 words
The method of claim 1 wherein said polymerase is able to utilize primers of 4 base pairs or more.
Claim 14
Dependent Claim
24 words
The method of claim 1 wherein said primer comprises 4-20 base pairs and said polymerase is able to utilize primers of 4-20 base pairs.
Claim 15
Dependent Claim
62 words
The method of claim 4 wherein said polymerase is non-discriminating for nucleotide analogs, and is a modified polymerase having less than 500 units of exonuclease activity per mg of polymerase said primer is single stranded RNA or DNA containing 4-10 bases and said polymerase is able to utilize primers of 4-10 bases, and said incubating comprises a pulse and a chase step.
Claim 16
Dependent Claim
14 words
The method of claim 1 wherein said primer is single stranded DNA or RNA.
Claim 17
Dependent Claim
33 words
The method of claim 1 wherein said annealing comprises heating said DNA molecule and said primer to above 65° C., and allowing the heated mixture to cool to 10° C. to 30° C.
Claim 18
Dependent Claim
15 words
The method of claim 1 wherein said incubating comprises a pulse and a chase step.
Claim 19
Dependent Claim
39 words
The method of claim 17 wherein said pulse step comprises mixing said annealed mixture with all four deoxynucleoside triphosphates and a processive DNA polymerase, wherein at least one said deoxynucleoside triphosphate is labelled and present in a limiting concentration.
Claim 20
Dependent Claim
19 words
The method of claim 18 wherein said pulse step incubation is carried out for 30 seconds to 20 minutes.
Claim 21
Dependent Claim
28 words
The method of claim 18 wherein said chase step comprises adding one said chain terminating agent to four separate aliquots of the mixture after performing said pulse step.
Claim 22
Dependent Claim
18 words
The method of claim 20 wherein said chase step incubation is carried out for 1 to 60 minutes.
Claim 23
Dependent Claim
12 words
The method of claim 1 wherein said terminating agent is a dideoxynucleotide.
Claim 24
Dependent Claim
17 words
The method of claim 1 wherein said terminating agent is a limiting level of one deoxynucleoside triphosphate.
Claim 25
Dependent Claim
16 words
The method of claim 1 wherein one said deoxynucleoside triphosphate is chosen from dITP or deazaguanosine.
Claim 26
Dependent Claim
15 words
The method of claim 1 wherein said primer is labelled prior to said annealing step.
Claim 27
Dependent Claim
12 words
The method of claim 25 wherein said incubating comprises a chase step.
Claim 28
Dependent Claim
11 words
The method of claim 25 wherein said primer is fluorescently labelled.
Claim 29
Dependent Claim
11 words
The method of claim 4 wherein said T7-type phage is T7.
Claim 30
Dependent Claim
15 words
The method of claim 12 wherein said mixture comprises T7 gene 2.5 or gene 4.
Claim 31
Dependent Claim
15 words
The method of claim 13 wherein said mixture comprises T7 gene 2.5 or gene 4.
Claim 32
Dependent Claim
81 words
A kit for DNA sequencing, comprising: a processive T7-type DNA polymerase, wherein said polymerase remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 500 units of exonuclease activity per mg of polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from (a) dITP and (b) a chain terminating agent.
Claim 33
Dependent Claim
81 words
A kit for DNA sequencing, comprising: a processive T7-type DNA polymerase, wherein said polymerase remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 500 units of exonuclease activity per mg of polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from (a) dITP and (b) a chain terminating agent.
Claim 34
Dependent Claim
30 words
The kit of claim 31 wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequencing reaction.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1992-01-03 | 1992-07-04 | 1993-01-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-01-03 | 1996-07-04 | 1997-01-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-01-03 | 2000-07-04 | 2001-01-03 | $7700.00 | Window Closed / Lapsed |