Method for determining the nucleotide base sequence of a DNA molecule
First Claim
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1. A method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of:
- annealing said DNA molecule with a labelled 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 DNA polymerase except reverse transcriptase, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein each agent terminates synthesis at a different nucleotide base, and wherein the concentration of all four deoxynucleoside triphosphates at the start of said incubating is sufficient to allow DNA synthesis to continue until terminated by said agent, andseparating 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.
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Abstract
This invention relates to T7-type DNA polymerases and methods for using them.
31 Citations
23 Claims
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1. 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 labelled 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 DNA polymerase except reverse transcriptase, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein each agent terminates synthesis at a different nucleotide base, and wherein the concentration of all four deoxynucleoside triphosphates at the start of said incubating is sufficient to allow DNA synthesis to continue until terminated by said agent, 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. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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2. 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 in a single vessel; incubating the annealed mixture with a DNA polymerase except reverse transcriptase in a single vessel containing four different deoxynucleoside triphosphates, at least one said deoxynucleoside triphosphate being labelled, under conditions in which said primer can be extended, incubating separate portions of the extended primer mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein each agent terminates synthesis at a different nucleotide base, and wherein said deoxynucleoside triphosphates are at a concentration sufficient to allow DNA synthesis to continue until terminated by said agent, 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.
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3. 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 labelled primer molecule able to hybridize to said DNA molecule. incubating the annealed mixture in the presence of four different deoxynucleoside triphosphates, a DNA polymerase except reverse tanscriptase, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein the concentration of all four deoxynucleoside triphosphates is sufficient to allow DNA synthesis to continue until terminated by said agent, and separating the DNA products of said incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined. - View Dependent Claims (23)
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4. 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 with a DNA polymerase except reverse transcriptase in the presence of four different deoxynucleoside triphosphates, at least one said deoxynucleoside triphosphate being labelled, under conditions in which said primer can be extended, incubating the extended primer mixture in the presence of four different deoxynucleoside triphosphates, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein said deoxynucleoside triphosphates are at a concentration sufficient to allow DNA synthesis to continue until terminated by said agent, 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.
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Specification