EP0351138A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
DNA sequencing
Assignee: Harvard University
•
Filing: 1989-07-06
•
Granted: —
•
Expires: 2009-07-06
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
EP
Data Source
google_patents
Last Synchronized
2026-11-07 14:37
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: 13
Claim 1
Independent Claim
129 words
A method for sequencing a strand of DNA comprising the steps of: providing said strand of DNA, annealing said strand with a primer able to hybridize to said strand, to give an annealed mixture, and incubating said annealed mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a first chain terminating agent, characterized in that said incubation is carried out under conditions in which said 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 quantity of each said first DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.
Claim 2
Independent Claim
129 words
A method for sequencing a strand of DNA comprising the steps of: providing said strand of DNA, annealing said strand with a primer able to hybridize to said strand, to give an annealed mixture, and incubating said annealed mixture with a deoxyribonucleoside triphosphate, a DNA polymerase, and a first chain terminating agent, characterized in that said incubation is carried out under conditions in which said 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 quantity of each said first DNA product being approximately the same for substantially all DNA products differing in length from 1 to 20 bases.
Claim 3
Dependent Claim
75 words
The method of claim 1 further including the steps of: separating said first DNA products by gel permeation according to molecular weight to form a first series of bands, each said first series band representing a said first DNA product of a particular molecular weight, wherein the intensity of each nearby first series band is approximately the same for substantially all said first series bands, and determining the position of each said first series band.
Claim 4
Dependent Claim
127 words
The method of claim 1 further characterized by providing one to three additional different chain terminating agents in said annealed mixture, each at a concentration different from that of every other chain terminating agent, wherein said DNA polymerase causes production of one to three additional series of additional DNA products, each DNA product of each additional series having one of said additional chain terminating agents at its elongated end, the quantity of each of said additional DNA product having the same chain terminating agent being approximately the same for all lengths within a range of 20 bases and being distinctly different from the quantity of all DNA products having a different chain terminating agent and being in a different series but having approximately the same molecular weight.
Claim 5
Dependent Claim
106 words
The method of claim 3 further including the steps of: separating all said DNA products by gel permeation according to molecular weight to form a total of two to four series of bands respectively, each said band of a series representing a DNA product having the same chain terminating agent as every other band in said series and being of a particular molecular weight, wherein the intensity of each nearby band within the same series is approximately the same and is distinctly different from the intensity of each nearby band of a different series, and determining the position and intensity of each band of each series.
Claim 6
Independent Claim
37 words
The method of any of the preceding claims further characterized in that said annealed mixture is provided with a manganese or iron ion, wherein said ion causes said polymerase to be non-discriminatory for said chain terminating agents.
Claim 7
Independent Claim
33 words
The method of any of the preceding claims further characterized in that said DNA polymerase is chosen from T7-type DNA polymerase, the large fragment of E. coli DNA polymerase I, and Taq polymerase.
Claim 8
Independent Claim
20 words
The method of any of the preceding claims further characterized in that said chain terminating agent is a dideoxynucleoside triphosphate
Claim 9
Independent Claim
20 words
The method of any of the preceding claims further characterized in that said annealed mixture is provided with a chelator.
Claim 10
Dependent Claim
29 words
A kit for use in sequencing DNA comprising a supply of a DNA polymerase and a chain terminating agent characterized in that it contains a manganese or iron ion.
Claim 11
Dependent Claim
29 words
A kit for use in sequencing DNA comprising a supply of a DNA polymerase and a chain terminating agent characterized in that it contains a manganese or iron ion.
Claim 12
Dependent Claim
28 words
The kit of claim 9 characterized in that said polymerase is a T7-type DNA polymerase, Klenow, or Taq polymerase and said chain terminating agent is a dideoxynucleoside triphosphate.
Claim 13
Dependent Claim
12 words
The kit of claim 9 characterized in that it contains a pyrophosphatase.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.