PH.phi.29 DNA polymerase
First Claim
1. 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 φ
29 type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase.
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Accused Products
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.
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Citations
20 Claims
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1. In a method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of:
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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 φ
29 type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A kit for DNA sequencing, comprising:
a φ
29-type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase, and a chain terminating agent.
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9. 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 φ - 29-type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase. - View Dependent Claims (10, 11, 12, 13, 14, 15)
- 29-type DNA polymerase or an exonuclease deficient φ
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16. A method for production of DNA molecules of greater than 10 kilobases in length comprising:
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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 φ
29-type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates.
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17. A method for amplification of a heterologous DNA molecule comprising the steps of:
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covalently bonding a φ
29-type terminal DNA sequence at one end of said DNA molecule to form a product; andincubating said product in the presence of a φ
29-type DNA polymerase, a terminal protein of a φ
29-type DNA polymerase or an exonuclease deficient φ
29-type DNA polymerase and a mixture of four different deoxynucleoside triphosphates. - View Dependent Claims (18, 19, 20)
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Specification