Patent US968850A successfully refreshed from Google Patents.
EP0265293A2
Expired / Lapsed
EP
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T7 DNA polymerase
Assignee: Harvard University
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Filing: 1987-12-24
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Granted: —
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Expires: 2007-12-24
Abstract
Abstract This invention relates to T7-type DNA polymerases and methods for using them including 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 with four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, said polymerase 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. The agent terminates at a different specific nucleotide base in each of the four vessels. The DNA products of the incubating reaction are separated according to their size so that at least part of the nucleotide base sequence of the . DNA molecule can be determined.
Inventors
- 👤 Stanley Tabor
- 👤 Charles C. Richardson
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 07:43
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: 70
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 DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, said polymerase 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 DNA polymerase, wherein said polymerase remains bound to said DNA molecule for at least 500 bases before dissociating in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, said polymerase 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
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 4
Dependent Claim
21 words
The method of claim 1 or claim 2 wherein said polymerase is that polymerase in cells infected with a T7-type phage.
Claim 5
Dependent Claim
21 words
The method of claim 4 wherein said T7-type phage is T7, T3, ΦI, ΦII, H, W31, gh-1, Y, A1122 or SP6.
Claim 6
Dependent Claim
11 words
The method of claim 4 wherein said T7-type phage is T-7.
Claim 7
Independent Claim
34 words
The method of any one of the preceding claims wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse reaction of a DNA sequencing reaction.
Claim 8
Independent Claim
18 words
The method of any one of the preceding claims wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.
Claim 9
Independent Claim
28 words
The method of any one of the preceding claims wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.
Claim 10
Dependent Claim
21 words
The method of claim 8 wherein said modified polymerase has less than 1 unit of exonuclease activity per mg of polymerase.
Claim 11
Dependent Claim
21 words
The method of claim 8 wherein said modified polymerase has less than 0.1 unit of exonuclease activity per mg of polymerase.
Claim 12
Independent Claim
18 words
The method of any one of claims 1 to 7 wherein said polymerase has no detectable exonuclease activity.
Claim 13
Independent Claim
23 words
The method of any one of the preceding claims wherein said polymerase is able to utilize primers of 10 base pairs or more.
Claim 14
Independent Claim
24 words
The method of any one of claims 1 to 11 wherein said polymerase is able to utilize primers of 4 base pairs or more.
Claim 15
Dependent Claim
24 words
The method of claim 13 wherein said primer comprises 4-20 base pairs and said polymerase is able to utilize primers of 4-20 base pairs.
Claim 16
Dependent Claim
18 words
The method of claim 13 or claim 14 wherein said mixture comprises T7 gene 2.5 or gene 4.
Claim 17
Dependent Claim
62 words
The method of claim 3 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 utilise primers of 4-10 bases, and said incubating comprises a pulse and a chase step.
Claim 18
Independent Claim
19 words
The method of any one of claims 1 to 15 wherein said primer is single stranded DNA or RNA.
Claim 19
Independent Claim
34 words
The method of any one of the preceding claims wherein said annealing comprises heating said DNA molecule and said primer to above 65°C, and allowing the heated mixture to cool to 0°C to 30°C.
Claim 20
Independent Claim
19 words
The method of any one of the preceding claims wherein said incubating comprises a pulse and a chase step.
Claim 21
Dependent Claim
39 words
The method of claim 19 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 22
Dependent Claim
19 words
The method of claim 20 wherein said pulse step incubation is carried out for 30 seconds to 20 minutes.
Claim 23
Dependent Claim
28 words
The method of claim 20 wherein said chase step comprises adding one said chain terminating agent to four separate aliquots of the mixture after performing said pulse step.
Claim 24
Dependent Claim
18 words
The method of claim 22 wherein said chase step incubation is carried out for 1 to 60 minutes.
Claim 25
Independent Claim
16 words
The method of any one of the preceding claims wherein said terminating agent is a dideoxynucleotide.
Claim 26
Independent Claim
21 words
The method of any one of the preceding claims wherein said terminating agent is a limiting level of one deoxynucleoside triphosphate.
Claim 27
Independent Claim
20 words
The method of any one of the preceding claims wherein one said deoxynucleoside triphosphate is chosen from dITP or deazaguanosine.
Claim 28
Independent Claim
20 words
The method of any one of claims 1 to 18 wherein said primer is labelled prior to said annealing step.
Claim 29
Dependent Claim
12 words
The method of claim 27 wherein the incubating comprises a chase step.
Claim 30
Dependent Claim
11 words
The method of claim 27 wherein said primer is fluorescently labelled.
Claim 31
Dependent Claim
80 words
A kit for DNA sequencing comprising: a processive 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 32
Dependent Claim
80 words
A kit for DNA sequencing comprising: a processive 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
10 words
The kit of claim 30 wherein said reagent is dITP.
Claim 34
Dependent Claim
32 words
The kit of claim 30 or claim 31 wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse reaction of DNA sequencing reaction.
Claim 35
Independent Claim
51 words
A method of producing active T7-type DNA polymerase from cloned fragments, comprising placing the genes encoding said polymerase under the control of non-leaky promoters in a single cell, inducing expression of said genes when said cell is in logarithmic growth phase, or stationary phase, and isolating said polymerase from said cell.
Claim 36
Independent Claim
51 words
A method of producing active T7-type DNA polymerase from cloned fragments, comprising placing the genes encoding said polymerase under the control of non-leaky promoters in a single cell, inducing expression of said genes when said cell is in logarithmic growth phase, or stationary phase, and isolating said polymerase from said cell.
Claim 37
Dependent Claim
23 words
The method of claim 33 wherein one of said genes is under the control of a promoter requiring T7 RNA polymerase for expression.
Claim 38
Dependent Claim
34 words
The method of claim 33 wherein said polymerase is active T7 DNA polymerase produced from two cloned genes, one said gene being under the control of a promoter requiring T7 RNA polymerase for expression.
Claim 39
Independent Claim
45 words
A method of purifying T7 DNA polymerase from cells comprising a vector from which said polymerase is expressed, comprising the steps of: lysing said cells, and passing said polymerase over a sizing column, a DE52 DEAE column, a phosphocellulose column, and a hydroxy apatite column.
Claim 40
Independent Claim
45 words
A method of purifying T7 DNA polymerase from cells comprising a vector from which said polymerase is expressed, comprising the steps of: lysing said cells, and passing said polymerase over a sizing column, a DE52 DEAE column, a phosphocellulose column, and a hydroxy apatite column.
Claim 41
Dependent Claim
20 words
The method of claim 36 wherein prior to said passing step said method comprises precipitating said polymerase with ammonium sulfate.
Claim 42
Dependent Claim
19 words
The method of claim 36 further comprising the steps of passing said polymerase over a Sephadex DEAE A-50 column.
Claim 43
Dependent Claim
14 words
The method of claim 36 wherein said sizing column is a Sephadex G150 column.
Claim 44
Independent Claim
32 words
A method of inactivating exonuclease activity in a DNA polymerase solution produced by recombinant DNA techniques, comprising: incubating said solution in a vessel containing oxygen, a reducing agent and a transition metal.
Claim 45
Independent Claim
32 words
A method of inactivating exonuclease activity in a DNA polymerase solution produced by recombinant DNA techniques, comprising: incubating said solution in a vessel containing oxygen, a reducing agent and a transition metal.
Claim 46
Dependent Claim
21 words
A gene encoding a T7-type DNA polymerase, said gene being genetically modified to reduce the activity of naturally occurring exonuclease activity.
Claim 47
Dependent Claim
21 words
A gene encoding a T7-type DNA polymerase, said gene being genetically modified to reduce the activity of naturally occurring exonuclease activity.
Claim 48
Dependent Claim
11 words
A T7-type DNA polymerase produced from the gene of claim 41.
Claim 49
Dependent Claim
10 words
The gene of claim 41 having a modified His residue.
Claim 50
Dependent Claim
14 words
The gene of claim 43, said His residue being His-123 of T7 DNA polymerase.
Claim 51
Dependent Claim
14 words
The gene of claim 43 or claim 44 wherein said His residue is deleted.
Claim 52
Dependent Claim
14 words
A T-7 type DNA polymerase produced from the gene of claim 44 or 45.
Claim 53
Dependent Claim
20 words
The gene of claim 41 wherein amino acids 118 to 123 are deleted from the gene encoding T7-type DNA polymerase.
Claim 54
Independent Claim
50 words
A method for producing blunt-ended double stranded DNA from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no 3' protruding.termini, comprising incubating said DNA molecule with a processive DNA polymerase essentially free from naturally occurring exonuclease activity.
Claim 55
Independent Claim
50 words
A method for producing blunt-ended double stranded DNA from a linear DNA molecule having a single stranded region, wherein the 3' end of said molecule is double stranded and has no 3' protruding.termini, comprising incubating said DNA molecule with a processive DNA polymerase essentially free from naturally occurring exonuclease activity.
Claim 56
Independent Claim
60 words
A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence.
Claim 57
Independent Claim
60 words
A method of amplification of a DNA sequence comprising annealing a first and second primer to opposite strands of a double stranded DNA sequence and incubating the annealed mixture with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, wherein said first and second primers anneal to opposite strands of said DNA sequence.
Claim 58
Dependent Claim
21 words
The method of claim 49 wherein said first and second primers have their 3' ends directed toward each other after annealing.
Claim 59
Dependent Claim
39 words
The method of claim 49 or claim 50 wherein said method further comprises, after said incubation step, denaturing the resulting DNA, annealing said first and second primers to said resulting DNA and incubating the annealed mixture with said polymerase.
Claim 60
Dependent Claim
20 words
The method of claim 51 wherein said cycle of denaturing, annealing and incubating is repeated from 10 to 40 times.
Claim 61
Independent Claim
23 words
The method of any one of claims 49 to 52 wherein said exonuclease activity is less than 1 unit per mg of polymerase.
Claim 62
Independent Claim
38 words
A method for labelling the 3' end of a DN A fragment comprising incubating said DNA fragment with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase and a labelled deoxynucleotide.
Claim 63
Independent Claim
38 words
A method for labelling the 3' end of a DN A fragment comprising incubating said DNA fragment with a processive DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase and a labelled deoxynucleotide.
Claim 64
Independent Claim
38 words
A method for in vitro mutagenesis of a cloned DNA fragment comprising providing a primer and a template, said primer and said template having a specific mismatched base, and extending said primer with a processive modified DNA polymerase.
Claim 65
Independent Claim
38 words
A method for in vitro mutagenesis of a cloned DNA fragment comprising providing a primer and a template, said primer and said template having a specific mismatched base, and extending said primer with a processive modified DNA polymerase.
Claim 66
Independent Claim
47 words
A method for in vitro mutagenesis of a cloned DNA fragment comprising providing said cloned fragment and synthesizing a DNA strand using a processive DNA polymerase, having less than 1 unit of exonuclease activity per mg of polymerase, under conditions which cause misincorporation of a nucleotide base.
Claim 67
Independent Claim
47 words
A method for in vitro mutagenesis of a cloned DNA fragment comprising providing said cloned fragment and synthesizing a DNA strand using a processive DNA polymerase, having less than 1 unit of exonuclease activity per mg of polymerase, under conditions which cause misincorporation of a nucleotide base.
Claim 68
Dependent Claim
36 words
A kit comprising a plurality of at least 25 oligomers suitable for DNA sequencing, each said oligomer being contained in a separate container and having a different base sequence of less than 15 bases in length.
Claim 69
Dependent Claim
36 words
A kit comprising a plurality of at least 25 oligomers suitable for DNA sequencing, each said oligomer being contained in a separate container and having a different base sequence of less than 15 bases in length.
Claim 70
Dependent Claim
14 words
The kit of claim 57 wherein said oligomers each comprise between 6-9 bases inclusive.
References Cited (Backward Citations)
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