DNA sequencing
First Claim
Patent Images
1. A method for sequencing a strand of DNA, comprising the steps of:
- providing said strand hybridized with a primer able to hybridize to said strand, to give an hybridized mixture,incubating said hydridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each said second DNA product having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
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Abstract
A method for sequencing a strand of DNA, including the steps of: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a chain terminating agent under conditions in which the polymerase causes the primer to be elongated to form a series of DNA products differing in length of the elongated primer, each DNA product having a chain terminating agent at its elongated end; the number of each DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.
97 Citations
34 Claims
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1. A method for sequencing a strand of DNA, comprising the steps of:
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providing said strand hybridized with a primer able to hybridize to said strand, to give an hybridized mixture, incubating said hydridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a first chain terminating agent, wherein said DNA polymerase causes said primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each said first DNA product having a said chain terminating agent at its elongated end, the number of molecules of each said first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases, and providing a second chain terminating agent in said hybridized mixture at a concentration different from said first chain terminating agent, wherein said DNA polymerase causes production of a second series of second DNA products differing in the length of the elongated primer, each said second DNA product having said second chain terminating agent at its elongated end, the number of molecules of each said second DNA products being approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and being distinctly different from the number of molecules of all said first DNA products having a length differing by no more than 20 bases from that of said second DNA products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 30, 31, 32)
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13. A method for sequencing a strand of DNA, comprising the steps of:
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providing a DNA polymerase, and incubating said polymerase and said strand of DNA in a solution comprising a manganese ion and a chain terminating agent wherein said ion is provided at a concentration effective for causing production of approximately equal numbers of molecules of these DNA products which differ in size from each other by no more than 20 bases. - View Dependent Claims (23, 28, 29)
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14. A kit for sequencing DNA comprising:
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a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising a manganese ion. - View Dependent Claims (16, 17, 18, 19, 20)
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15. A kit for sequencing DNA comprising:
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a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising an iron ion.
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21. A method for sequencing DNA comprising:
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providing a DNA polymerase in a reaction medium in which said DNA polymerase is substantially non-discriminating for a chain terminating agent, and incubating said DNA in said reaction medium with said DNa polymerase, a primer, four deoxyribonucleosie triphosphates and a chain terminating agent to elongate said primer to form a series of DNA products differing in the length of the elongated primer and having said chain terminating agent at the elongated end, the number of molecules of each said DNA product being approximately the same for substantially all those DNA products differing in size by no more than 20 bases. - View Dependent Claims (22)
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24. A method for sequencing a strand of DNA, comprising the steps of:
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providing said strand hybridized with a primer able to hybridize to said strand, to give a hybridized mixture, and incubating said hybridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a plurality of chain terminating agents in a reaction medium in which said polymerase causes said primer to be elongated to form a series of DNA products differing in the length of the elongated primer, each DNA product having a first said chain terminating agent at its elongated end, wherein the number of molecules of said DNA products is approximately the same for substantially all DNA products differing in length by no more than 20 bases. - View Dependent Claims (25, 26, 27)
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33. A method for sequencing a strand of DNA, comprising the steps of:
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providing a DNA polymerase, and incubating said polymerase and said strand of DNA in a solution comprising an iron ion and a chain terminating agent wherein said ion is provided at a concentration effective for causing production of approximately equal numbers of molecules of DNA products differing in size by no more than 20 bases.
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34. A method for sequencing a strand of DNA, comprising the steps of:
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providing said strand hybridized with a primer able to hybridize to said strand to give an hybridized mixture, incubating said hybridized mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and a plurality of different chain terminating agents each capable of terminating the DNa synthesis at a different nucleotide base, to cause said primer to be elongated to form a series of DNA products differing in length with each DNA product having a chain terminating agent at its elongated end, the amount of each said chain terminating agent being distinctly different from that of each other chain terminating agent, and said incubation causing the total amount of said series terminating with one chain terminating agent to be distinctly different from the total amount of said series terminating with each different chain terminating agent, and separating said series according to length, whereby the DNA products of approximately the same length containing one chain terminating agent are distinctly different in amount from those containing any other chain terminating agent.
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Specification