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US4962020A
Expired / Lapsed
US
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DNA sequencing
Assignee: Harvard University
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Filing: 1988-07-12
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Granted: 1990-10-09
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Expires: 2008-07-12
Abstract
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.
Inventors
- 👤 Stanley Tabor
- 👤 Charles C. Richardson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-10 01:49
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6869; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/81; Y10S436/00; Y10S436/807; Y10S436/808; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 42
Claim 1
Independent Claim
239 words
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.
Claim 2
Independent Claim
239 words
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.
Claim 3
Dependent Claim
130 words
The method of claim 1 further including the steps of: separating said first and second DNA products by gel permeation according to molecular weight to form a first and second series of bands respectively, each said first or second series band representing a said first or second DNA product, respectively, of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, and wherein the intensity of substantially all nearby second series bands is approximately the same, and wherein the intensity of substantially all bands of the first series is distinctly different from the intensity of each nearby band of the second series, and determining the position and intensity of each said first and second series band.
Claim 4
Dependent Claim
148 words
The method of claim 1 further comprising the step of: providing a third chain terminating agent in said hybridized mixture at a concentration different from said first and second chain terminating agents, wherein said polymerase causes production of a third series of third DNA products differing in the length of the elongated primer, each said third DNA product having said third chain terminating agent at its elongated end, the number of molecules of each said third DNA product being approximately the same for substantially all said third DNA products which differ in length by no more than 20 bases, wherein the numbers of molecules of substantially all said first, all said second and all said third DNA products are distinctly different from the numbers of molecules of all those DNA products of a different series which are of lengths differing from them by no more than 20 bases.
Claim 5
Dependent Claim
132 words
The method of claim 3 further including the steps of: separating said first, second and third DNA products by gel permeation according to molecular weight to form a first, second and third series of bands, respectively, each said first, second or third series band representing a said first, second or third DNA product, respectively, of a given molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, wherein the intensity of substantially all nearby second series bands is approximately the same, and the intensity of substantially all nearby third series bands is approximately the same, and wherein the intensity of substantially all nearby bands of different series is distinctly different, and determining the position and intensity of each said band.
Claim 6
Dependent Claim
152 words
The method of claim 3 further comprising the step of: providing in said hybridized mixture a fourth chain terminating agent at a concentration different from said first, second and third chain terminating agents, wherein said DNA polymerase causes production of a fourth series of fourth DNA products differing in the length of the elongated primer, each said fourth DNA product having said fourth chain terminating agent at its elongated end, the number of molecules of each said fourth DNa product being approximately the same or substantially all fourth DNa products differing in length by no more than 20 bases, wherein the number of molecules of substantially all said first, all said second, all said third, and all said fourth series of DNA products is distinctly different from all the number of molecules of those DNa products of a different series which differ in length from them by no more than 20 bases.
Claim 7
Dependent Claim
137 words
The method of claim 5, further including the steps of: separating said first, second, third and fourth DNA products by gel permeation according to molecular weight to form a first, second third and fourth series of bands respectively, each said first, second, third and fourth series band representing a said first, second, third or fourth DNA product of a given molecular weight, wherein each said second, third and fourth series produce second, third and fourth bands, wherein the intensity of substantially all nearby first series bands, or substantially all nearby second series bands, or substantially all nearby third series bands, or substantially all nearby fourth series bands is approximately the same, and wherein the intensity of substantially all nearby bands in a different series is distinctly different, and determining the position and intensity of each said band.
Claim 8
Dependent Claim
38 words
The method of claim 1, 2, 3, 4, 5 or 6 wherein said hybridized mixture is provided with a manganese or iron ion, wherein said ion causes said polymerase to be non-discriminatory for a said chain terminating agent.
Claim 9
Dependent Claim
23 words
The method of claim 5 wherein said DNA products are separated according to molecular weight in less than four lanes of a gel.
Claim 10
Dependent Claim
18 words
The method of claim 8 wherein the intensity of each band is measured by a gel reading apparatus.
Claim 11
Dependent Claim
21 words
The method of claim 1, 2, 3, 4, 5, or 6 wherein each said chain terminating agent is a dideoxynucleoside triphosphate.
Claim 12
Dependent Claim
14 words
The method of claim 7, wherein said hybridized mixture is provided with a chelator.
Claim 13
Dependent Claim
11 words
The method of claim 11, said chelator being citrate or isocitrate.
Claim 14
Independent Claim
72 words
A method for sequencing a strand of DNA, comprising the steps of: 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.
Claim 15
Independent Claim
72 words
A method for sequencing a strand of DNA, comprising the steps of: 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.
Claim 16
Dependent Claim
28 words
A kit for sequencing DNA comprising: a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising a manganese ion.
Claim 17
Dependent Claim
28 words
A kit for sequencing DNA comprising: a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising a manganese ion.
Claim 18
Dependent Claim
28 words
A kit for sequencing DNA comprising: a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising an iron ion.
Claim 19
Dependent Claim
28 words
A kit for sequencing DNA comprising: a first container comprising a DNA polymerase, a second container comprising a chain terminating agent, and a compound comprising an iron ion.
Claim 20
Dependent Claim
23 words
The kit of claim 14 or 15 wherein said polymerase is a T7-type DNA polymerase, suitable for DNA sequencing, Klenow, or Taq polymerase.
Claim 21
Dependent Claim
18 words
The kit of claim 14, 16, or 15, wherein each said chain terminating agent is a dideoxynucleoside triphosphate.
Claim 22
Dependent Claim
10 words
The kit of claim 16 further comprising a deoxyribonucleoside triphosphate.
Claim 23
Dependent Claim
9 words
The kit of claim 14 further comprising a chelator.
Claim 24
Dependent Claim
11 words
The kit of claim 19, said chelator being citrate or isocitrate.
Claim 25
Independent Claim
106 words
A method for sequencing DNA comprising: 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.
Claim 26
Independent Claim
106 words
A method for sequencing DNA comprising: 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.
Claim 27
Dependent Claim
21 words
The method of claim 21 wherein said DNA polymerase is a T7-type DNA polymerase provided in a solution comprising manganese ions.
Claim 28
Independent Claim
22 words
The method of claims 7, 13, or 22 wherein said manganese ions are at a concentration between 5 μM and 20 μM.
Claim 29
Independent Claim
119 words
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 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.
Claim 30
Independent Claim
119 words
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 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.
Claim 31
Dependent Claim
12 words
The method of claim 24, wherein said reaction medium comprises manganese ions.
Claim 32
Dependent Claim
19 words
The method of claim 25, wherein said manganese ions are at a concentration between 5 μM and 20 mM.
Claim 33
Dependent Claim
23 words
The method of claim 25, wherein said reaction medium comprises a chelator and manganese ions at a concentration between 0.005 and 100 mM.
Claim 34
Dependent Claim
24 words
The method of claim 13 wherein said solution comprises a chelator and said manganese ion is at a concentration between 0.005 nd 100 mM.
Claim 35
Dependent Claim
19 words
The method of claim 13 wherein said manganese ion is at a concentration between 5 μM and 20 mM.
Claim 36
Dependent Claim
36 words
The method of claim 1, 2, 3, 4, 5 or 6, wherein said hybridized mixture is provided with a manganese ion, wherein said ion causes said polymerase to be non-discriminating for a said chain terminating agent.
Claim 37
Dependent Claim
34 words
The method of claim 1, 2, 3, 4, 5 or 6, wherein said DNA polymerase is selected from the group consisting of the large fragment of E. coli DNA polymerase I and Taq polymerase.
Claim 38
Dependent Claim
20 words
The method of claim 1, 2, 3, 4, 5 or 6, wherein said DNA polymerase is a T7-type DNA polymerase.
Claim 39
Independent Claim
67 words
A method for sequencing a strand of DNA, comprising the steps of: 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.
Claim 40
Independent Claim
67 words
A method for sequencing a strand of DNA, comprising the steps of: 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.
Claim 41
Independent Claim
178 words
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 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.
Claim 42
Independent Claim
178 words
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 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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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1993-10-08 | 1994-04-09 | 1994-10-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-10-08 | 1998-04-09 | 1998-10-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-10-08 | 2002-04-09 | 2002-10-09 | $7700.00 | Window Closed / Lapsed |