US5075216A
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Methods for dna sequencing with thermus aquaticus dna polymerase
Assignee: Roche Molecular Systems Inc
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Filing: 1988-09-23
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Granted: 1991-12-24
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Expires: 2008-09-23
Abstract
Abstract Dideoxynucleotide DNA sequencing methods can be dramatically improved by utilizing the DNA polymerase from Thermus aquaticus to catalyze the primer extension reactions.
Inventors
- 👤 Michael A. Innis
- 👤 Kenneth B. Myambo
- 👤 David H. Gelfand
- 👤 Mary Ann D. Brow
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 22:29
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/70; C12Q1/701; C12Q1/68; C12Q1/6844; C12Q1/686; C12Q1/6869; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 33
Claim 1
Independent Claim
71 words
In a method for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside- 5'-triphosphate chain termination procedure, wherein said sequence is determined by extending an oligonucleotide primer in a templatedependent manner in the presence of an agent for polymerization, four dideoxynucleoside- 5'-triphosphates (dNTPs), and a dideoxynucleoside- 5'-triphosphate (ddNTP), the improvement comprising extending said primer in the presence of the agent for polymerization that is Thermus aquaticus DNA polymerase.
Claim 2
Dependent Claim
71 words
In a method for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside- 5'-triphosphate chain termination procedure, wherein said sequence is determined by extending an oligonucleotide primer in a templatedependent manner in the presence of an agent for polymerization, four dideoxynucleoside- 5'-triphosphates (dNTPs), and a dideoxynucleoside- 5'-triphosphate (ddNTP), the improvement comprising extending said primer in the presence of the agent for polymerization that is Thermus aquaticus DNA polymerase.
Claim 3
Dependent Claim
10 words
The method of claim 1, wherein said primer is labeled.
Claim 4
Dependent Claim
15 words
The method of claim 1, wherein one of the four dNTPs or ddNTP is labeled.
Claim 5
Dependent Claim
15 words
The method of claim 1, wherein the four dideoxynucleoside-5'-triphosphates are dATP, dCTP, dGTP, and TTP.
Claim 6
Dependent Claim
18 words
The method of claim 1, wherein the four dideoxynucleoside- 5'-triphosphates are dATP, dCTP, c 7 dGTP, and TTP.
Claim 7
Dependent Claim
16 words
The method of claim 1, wherein the four dideoxynucleoside- 5'-triphosphates are dATP, dCTP, dITP, and TTP.
Claim 8
Dependent Claim
18 words
The method of claim 1, wherein said nucleic acid segment was produced by an asymmertric polymerase chain reaction.
Claim 9
Dependent Claim
18 words
The method of claim 1, wherein said nucleic acid segment was produced by an asymmetric polymerase chain reaction.
Claim 10
Dependent Claim
14 words
The method of claim 1, wherein no KCl is present in the reaction mixture.
Claim 11
Dependent Claim
24 words
The method of claim 1, wherein the DNA polymerase is present in up to a 2.5 -fold molar excess over the nucleic acid segment.
Claim 12
Dependent Claim
19 words
The method of claim 2, wherein said primer is labeled with 32 P, 35 S, or a fluorescent molecule.
Claim 13
Dependent Claim
21 words
The method of claim 3, wherein said dNTP or ddNTP is labeled with 35 S, 32 P, or a fluorescent molecule.
Claim 14
Dependent Claim
51 words
The method of claim 3, wherein said extension reaction is carried out first at a low temperature and in the presence of three unlabeled dNTPs and one labeled dNTP each present at a concentration of less than 1 μM and then at higher temperatures in higher concentrations of the unlabeled dNTPs.
Claim 15
Dependent Claim
11 words
The method of claim 4, wherein said dideoxynucleoside- 5'-triphosphate is ddATP.
Claim 16
Dependent Claim
11 words
The method of claim 4, wherein said dideoxynucleoside- 5'-triphosphate is ddCTP.
Claim 17
Dependent Claim
11 words
The method of claim 4, wherein said dideoxynucleoside- 5'-triphosphate is ddGTP.
Claim 18
Dependent Claim
11 words
The method of claim 4, wherein said dideoxynucleoside- 5'-triphosphate is ddTTP.
Claim 19
Dependent Claim
15 words
The method of claim 4, wherein c 7 dGTP is also present during primer extension.
Claim 20
Dependent Claim
19 words
The method of claim 4, wherein each dNTP is present at a concentration of 5 μM to 30 μM.
Claim 21
Dependent Claim
31 words
The method of claim 13, wherein the concentration of said labeled dNTP is 0.5 μM and the concentration of each unlabeled dNTP is 1.0 μM during said low temperature extension reaction.
Claim 22
Dependent Claim
11 words
The method of claim 14, wherein the dATP:ddATP ratio is 1:32.
Claim 23
Dependent Claim
11 words
The method of claim 15, wherein the dCTP:ddCTP ratio is 1:16.
Claim 24
Dependent Claim
11 words
The method of claim 16, wherein the dGTP:ddGTP ratio is 1:6.
Claim 25
Dependent Claim
11 words
The method of claim 17, wherein the TTP:ddTTP ratio is 1:48.
Claim 26
Dependent Claim
14 words
The method of claim 19, wherein the concentration of each dNTP is 10 μM.
Claim 27
Dependent Claim
54 words
A kit for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside-5'-triphosphate chain termination procedure, which kit comprises (a) a primer for extending, in a template-dependent manner, a nucleic acid comprising a sequence complementary to said nucleic acid segment; (b) four deoxyribonucleoside-5'-triphosphates (dNTPs); (c) four dideoxyribonucleoside-5'-triphosphates (ddNTPs); and (d) Taq polymerase.
Claim 28
Dependent Claim
54 words
A kit for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside-5'-triphosphate chain termination procedure, which kit comprises (a) a primer for extending, in a template-dependent manner, a nucleic acid comprising a sequence complementary to said nucleic acid segment; (b) four deoxyribonucleoside-5'-triphosphates (dNTPs); (c) four dideoxyribonucleoside-5'-triphosphates (ddNTPs); and (d) Taq polymerase.
Claim 29
Dependent Claim
23 words
The kit of claim 26, wherein said dNTPs are dGTP, dATP, TTP, and dCTP, and said ddNTPs are ddGTP, ddATP, ddTTP, and ddCTP.
Claim 30
Dependent Claim
10 words
The kit of claim 26 that comprises c 7 dGTP.
Claim 31
Dependent Claim
40 words
The kit of claim 26, wherein said dNTPs are described as a set of four dNTPs selected from the group consisting of: dGTP, dATP, dCTP, and TTP; c 7 dGTP, dATP, dCTP, and TTP; and dITP, dATP, dCTP, and TTP.
Claim 32
Dependent Claim
26 words
The kit of claim 26, wherein said primer hybridizes to an M13 cloning vector and is suitable for sequencing cloned inserts in M13 and/or pUC-based vectors.
Claim 33
Dependent Claim
52 words
The kit of claim 27, that comprises: (i) a G-termination mix comprising dGTP, dATP, TTP, dCTP, and ddGTP; (ii) a A-termination mix comprising dGTP, dATP, TTP, dCTP, and ddATP; (iii) a T-termination mix comprising dGTP, dATP, TTP, dCTP, and ddTTP; and (iv) a C-termination mix comprising dGTP, dATP, TTP, dCTP, and ddCTP.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-12-23 | 1995-06-24 | 1995-12-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-12-23 | 1999-06-24 | 1999-12-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-12-23 | 2003-06-24 | 2003-12-24 | $7700.00 | Window Closed / Lapsed |