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US5001050A
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PHφ29 DNA polymerase
Assignee: Consejo Superior de Investigaciones Científicas
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Filing: 1989-03-24
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Granted: 1991-03-19
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Expires: 2009-03-24
Abstract
Abstract An improved method for determining the nucleotide base sequence of a DNA molecule. The method includes annealing the DNA molecule with a primer molecule able to hybridize to the DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each the agent terminates DNA synthesis at a different nucleotide base; and separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA can be determined. The improvement is provision of a DNA-polymerase which is a φ29-type DNA polymerase.
Inventors
- 👤 Luis Blanco
- 👤 Antonio Bernad
- 👤 Margarita Salas
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 14:50
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; C12N; C12N9/00; C12N9/10; C12N9/12; C12N9/1241; C12N9/1252; C12Q1/6844; C12Q1/686; C12Q1/6865; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
123 words
In a method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more 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 the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of said DNA can be determined, the improvement wherein said DNA polymerase comprises a φ29 type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase.
Claim 2
Dependent Claim
123 words
In a method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule; incubating the annealed mixture in a vessel containing four different deoxynucleoside triphosphates, a DNA polymerase, and one or more 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 the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of said DNA can be determined, the improvement wherein said DNA polymerase comprises a φ29 type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase.
Claim 3
Dependent Claim
22 words
The method of claim 1 wherein said φ29-type DNA polymerase is that phage DNA polymerase in cells infected with a φ29-type phage.
Claim 4
Dependent Claim
41 words
The method of claim 1 wherein said φ29-type DNA polymerase is selected from the DNA polymerases of the phage group consisting of: φ29, Cp-1, PRD1, φ15, φ21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, and L17.
Claim 5
Dependent Claim
27 words
The method of claim 1 wherein said φ29-type DNA polymease is a modified polymerase having less than ten percent of the exonuclease activity of the naturally-occurring polymerase.
Claim 6
Dependent Claim
21 words
The method of claim 4 wherein said polymerase is modified to have less than one percent of the naturally-occurring exonuclease activity.
Claim 7
Dependent Claim
15 words
The method of claim 5 wherein said φ29-type DNA polymerase has substantially no exonuclease activity.
Claim 8
Dependent Claim
13 words
The method of claim 1 wherein said terminating agent is a dideoxynucleoside triphosphate.
Claim 9
Dependent Claim
22 words
A kit for DNA sequencing, comprising: a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase, and a chain terminating agent.
Claim 10
Dependent Claim
22 words
A kit for DNA sequencing, comprising: a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase, and a chain terminating agent.
Claim 11
Dependent Claim
52 words
In a method for 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 DNA polymerase, the improvement comprising employing as said DNA polymerase a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase.
Claim 12
Dependent Claim
52 words
In a method for 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 DNA polymerase, the improvement comprising employing as said DNA polymerase a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase.
Claim 13
Dependent Claim
21 words
The method of claim 9 wherein said first and second primers have their 3' ends directed towards each other after annealing.
Claim 14
Dependent Claim
38 words
The method of claim 9 wherein said method further comprises, after said incubation step, denaturing the resulting DNA, annealing said first and second primers to the denatured DNA and incubating the last said annealed mixture with said polymerase.
Claim 15
Dependent Claim
18 words
The method of claim 11 wherein said cycle of denaturing, annealing, and incubating is repeated from 10-40 times.
Claim 16
Dependent Claim
41 words
The method of claim 9 wherein said φ29-type DNA polymerase is selected from the DNA polymerases of the phage group consisting of: φ29, Cp-1, PRD1, φ15, φ21, PZE, PZA, Nf, M2Y, B103, SF5, GA-1, Cp-5, Cp-7, PR4, PR5, PR722, and L17.
Claim 17
Dependent Claim
23 words
The method of claim 9 wherein said polymerase exhibits less than 10% of the naturally-occurring exonuclease activity exhibited by the corresponding naturally-occurring polymerase.
Claim 18
Dependent Claim
14 words
The method of claim 9 wherein said DNA polymerase has no detectable exonuclease activity.
Claim 19
Independent Claim
58 words
A method for production of DNA molecules of greater than 10 kilobases in length comprising: providing a template DNA molecule; annealing a primer with said template molecule; and incubating the annealed primer and template molecules in the presence of a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates.
Claim 20
Independent Claim
58 words
A method for production of DNA molecules of greater than 10 kilobases in length comprising: providing a template DNA molecule; annealing a primer with said template molecule; and incubating the annealed primer and template molecules in the presence of a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates.
Claim 21
Independent Claim
66 words
A method for amplification of a heterologous DNA molecule comprising the steps of: covalently bonding a φ29-type terminal DNA sequence at one end of said DNA molecule to form a product; and incubating said product in the presence of a φ29-type DNA polymerase, a terminal protein of a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates.
Claim 22
Independent Claim
66 words
A method for amplification of a heterologous DNA molecule comprising the steps of: covalently bonding a φ29-type terminal DNA sequence at one end of said DNA molecule to form a product; and incubating said product in the presence of a φ29-type DNA polymerase, a terminal protein of a φ29-type DNA polymerase or an exonuclease deficient φ29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates.
Claim 23
Dependent Claim
22 words
The method of claim 17 further comprising the step of providing a φ29-type terminal sequence at each end of said DNA molecule.
Claim 24
Dependent Claim
20 words
The method of claim 18 wherein said terminal sequence is provided on a DNA fragment of less than 500 nucleotides.
Claim 25
Dependent Claim
25 words
The method of claim 17 wherein said terminal protein is the terminal protein of the φ29-type phage in which the φ29-type DNA polymerase naturally occurs.
References Cited (Backward Citations)
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