US5976802A
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Simultaneous sequencing of nucleic acids
Assignee: European Molecular Biology Laboratory
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Filing: 1996-04-26
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Granted: 1999-11-02
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Expires: 2016-04-26
Abstract
Abstract The invention concerns a method for the sequence-specific labelling of nucleic acids comprising the generation of labelled nucleic acid fragments by an enzymatic labelling reaction in which a labelled deoxyribonucleoside triphosphate is attached to a nucleic acid primer molecule and the nucleic acid sequence is determined by means of the label, wherein the labelling reaction is carried out in a single reaction vessel with the simultaneous presence of one or several nucleic acid primer molecules and at least two labelled deoxyribonucleoside triphosphates which each contain different labelling groups and different bases and under conditions in which only one single type of labelled deoxyribonucleoside triphosphate can be attached to a nucleic acid primer molecule.
Inventors
- 👤 Wilhelm Ansorge
- 👤 Hartmut Voss
- 👤 Josef Stegemann
- 👤 Stefan Wiemann
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 05:45
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/6869
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
148 words
A method for the simultaneous and specific labeling of nucleic acids comprising: a) providing an enzymatic labelling reaction in a single reaction vessel, said reaction vessel comprising: i) one or more target nucleic acid molecules; ii) at least one nucleic acid primer molecule of a distinct sequence, wherein each primer molecule hybridizes to a distinct area of the target molecule or molecules; and iii) a first labelled 3' unblocked deoxyribonucleotide triphosphate of a particular base, and a second labelled 3' unblocked deoxyribonucleotide triphosphate of a different base, wherein the first labelled deoxyribonucleotide triphosphate contains a different label than the second deoxyribonucleotide triphosphate; b) hybridizing at least one primer molecule to the target molecule or molecules; c) attaching at least one of the labeled deoxyribonucleoside triphosphates to at least one primer molecule; and d) detecting the labeled deoxyribonucleotide triphosphates that are now attached to at least one primer molecule.
Claim 2
Independent Claim
148 words
A method for the simultaneous and specific labeling of nucleic acids comprising: a) providing an enzymatic labelling reaction in a single reaction vessel, said reaction vessel comprising: i) one or more target nucleic acid molecules; ii) at least one nucleic acid primer molecule of a distinct sequence, wherein each primer molecule hybridizes to a distinct area of the target molecule or molecules; and iii) a first labelled 3' unblocked deoxyribonucleotide triphosphate of a particular base, and a second labelled 3' unblocked deoxyribonucleotide triphosphate of a different base, wherein the first labelled deoxyribonucleotide triphosphate contains a different label than the second deoxyribonucleotide triphosphate; b) hybridizing at least one primer molecule to the target molecule or molecules; c) attaching at least one of the labeled deoxyribonucleoside triphosphates to at least one primer molecule; and d) detecting the labeled deoxyribonucleotide triphosphates that are now attached to at least one primer molecule.
Claim 3
Dependent Claim
188 words
The method of claim 1 comprising: a) providing an enzymatic labelling reaction in a single reaction vessel, said reaction vessel comprising: i) one or more target nucleic acid molecules; ii) a first nucleic acid primer molecule of a distinct sequence, and a second nucleic acid primer molecule of a second distinct sequence, wherein the first primer molecule and the second primer molecule hybridize to different and distinct areas of the target molecule or molecules; and iii) a first labelled 3' unblocked deoxyribonucleotide triphosphate of a particular base, and a second labelled 3' unblocked deoxyribonucleotide triphosphate of a different base, wherein the first labelled deoxyribonucleotide triphosphate contains a different label than the second deoxyribonucleotide triphosphate; b) hybridizing the first and second primer molecules to the target molecule or molecules; c) attaching the labeled deoxyribonucleoside triphosphates to the first and second primer molecules, under conditions in which the first labelled deoxyribonucleoside triphosphate is attached to the first primer molecule, and the second labeled deoxyribonucleoside triphosphate is attached to the second primer molecule; and d) detecting the labeled deoxyribonucleotide triphosphates that are now attached to the first and second primer molecules.
Claim 4
Dependent Claim
26 words
The method of claim 1 wherein the labeled 3' unblocked deoxyribonucleoside triphosphate is attached directly to the 3' end of each primer molecule at position +1.
Claim 5
Dependent Claim
27 words
The method of claim 1 wherein a DNA polymerase without 3' exonuclease activity is used to attach the labeled 3' unblocked deoxyribonucleotide triphosphates to the primer molecules.
Claim 6
Dependent Claim
13 words
The method of claim 4 wherein a modified T7 DNA polymerase is used.
Claim 7
Dependent Claim
27 words
The method of claim 1 wherein a DNA polymerase with 3' exonuclease activity is used to attach the labeled 3' unblocked deoxyribonucleoside triphosphates to the primer molecules.
Claim 8
Dependent Claim
40 words
The method of claim 1 wherein the labeled 3' unblocked deoxyribonucleoside triphosphate is in inserted in a substitution reaction as the last base of the primer molecule at position -1, by sequential exonuclease and polymerase activities of the DNA polymerase.
Claim 9
Dependent Claim
14 words
The method of claim 1 wherein the primer molecules do not contain labeling groups.
Claim 10
Dependent Claim
39 words
The method of claim 1 wherein the primer molecules do contain labeling groups, and the labeling groups on the primer molecules are not the same as the labeling groups on the first or second labeled 3' unblocked deoxyribonucleoside triphosphates.
Claim 11
Dependent Claim
26 words
The method of claim 1 wherein the labeling group on the primer molecules are selected from the group consisting of fluorescent dyes, metals, biotin and digoxigenin.
Claim 12
Dependent Claim
22 words
The method of claim 1 wherein the labeled 3' unblocked deoxyribonucleoside triphosphates are used at a concentration of 0.05 to 5 μmol/liter.
Claim 13
Dependent Claim
22 words
The method of claim 11 wherein the labeled 3' unblocked deoxyribonucleotide triphosphates are used at a concentration of 0.1 to 2.5 μmol/liter.
Claim 14
Dependent Claim
55 words
The method of claim 1 wherein two different Drimer molecules are used and wherein the primer molecules are selected in such a way that the base to be attached at the 3' end of the first primer molecule is different from the base to be attached at the 3' end of the second primer molecule.
Claim 15
Dependent Claim
50 words
The method of claim 1 wherein two different primer molecules are used and wherein the base to be attached at the 3' end of the first primer molecule at position +1 is not the same as the base that could be attached as position +2 of the second primer molecule.
Claim 16
Dependent Claim
50 words
The method of claim 1 wherein two different Drimer molecules are used and wherein the base to be attached at the 3' end of the first primer molecule at position +1 is not the same as the base that could be attached at position +1 of the second primer molecule.
Claim 17
Dependent Claim
20 words
The method of claim 1 wherein the first and the second 3' unblocked deoxyribonucleoside triphosphates contain different fluorescent labeling groups.
Claim 18
Dependent Claim
47 words
The method of claim 16 wherein the fluorescent labeling groups are selected from the group consisting of fluorescein or derivatives thereof, Texas red, rhodamine or derivatives thereof, phycoerythrin, CY3, CY5, CY2, CY7, coumarin, infrared 40, MR 200, bodipy dyes, IRD 40 and 1,N 6 -ethano modification groups.
Claim 19
Dependent Claim
15 words
The method of claim 16 wherein the different fluorescent labeling groups can be detected simultaneously.
Claim 20
Dependent Claim
15 words
The method of claim 16 wherein two or more different lasers are used for detection.
Claim 21
Dependent Claim
12 words
The method of claim 16 wherein a laser is used for detection.
Claim 22
Independent Claim
233 words
A method for the simultaneous sequencing of nucleic acids comprising: a) providing an enzymatic labelling reaction in a single reaction vessel, said reaction vessel comprising: i) one or more target nucleotide molecules; ii) a first nucleic acid primer molecule of a distinct sequence, and a second nucleic acid primer molecule of a second distinct sequence, wherein the first primer molecule and the second primer molecule hybridize to different and distinct areas of the target molecule or molecules; and iii) a first labelled deoxyribonucleotide triphosphate of a particular base, and a second labelled deoxyribonucleotide triphosphate of a different base, wherein the first labelled deoxyribonucleotide triphosphate contains a different label than the second deoxyribonucleotide triphosphate; b) hybridizing the first and second primer molecules to the target molecule or molecules; c) attaching the labeled deoxyribonucleoside triphosphates to the first and second primer molecules, under conditions in which the first labelled deoxyribonucleoside triphosphate is attached to the first primer molecule, and the second labeled deoxyribonucleoside triphosphate is attached to the second primer molecule; d) dividing the reaction mixture from the single vessel into separate vessels, and adding an extension reagent, wherein each vessel contains four unlabeled deoxyribonucleoside triphosphates and, in each separate vessel, a different chain termination molecule, e) separating the nucleic acid fragments formed in d); and f) detecting the labels on the first and second deoxyribonucleotide triphosphates and determining the sequence of the target molecules.
Claim 23
Independent Claim
233 words
A method for the simultaneous sequencing of nucleic acids comprising: a) providing an enzymatic labelling reaction in a single reaction vessel, said reaction vessel comprising: i) one or more target nucleotide molecules; ii) a first nucleic acid primer molecule of a distinct sequence, and a second nucleic acid primer molecule of a second distinct sequence, wherein the first primer molecule and the second primer molecule hybridize to different and distinct areas of the target molecule or molecules; and iii) a first labelled deoxyribonucleotide triphosphate of a particular base, and a second labelled deoxyribonucleotide triphosphate of a different base, wherein the first labelled deoxyribonucleotide triphosphate contains a different label than the second deoxyribonucleotide triphosphate; b) hybridizing the first and second primer molecules to the target molecule or molecules; c) attaching the labeled deoxyribonucleoside triphosphates to the first and second primer molecules, under conditions in which the first labelled deoxyribonucleoside triphosphate is attached to the first primer molecule, and the second labeled deoxyribonucleoside triphosphate is attached to the second primer molecule; d) dividing the reaction mixture from the single vessel into separate vessels, and adding an extension reagent, wherein each vessel contains four unlabeled deoxyribonucleoside triphosphates and, in each separate vessel, a different chain termination molecule, e) separating the nucleic acid fragments formed in d); and f) detecting the labels on the first and second deoxyribonucleotide triphosphates and determining the sequence of the target molecules.
Claim 24
Dependent Claim
17 words
The method of claim 21 wherein the deoxyribonucleoside triphosphates are used at a concentration of 0.05-5 mmol/liter.
Claim 25
Dependent Claim
17 words
The method of claim 22 wherein the deoxyribonucleoside triphosphates are used at a concentration of 0.1-2.5 mmol/liter.
References Cited (Backward Citations)
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| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 2002-11-01 | 2003-05-03 | 2003-11-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2006-11-01 | 2007-05-03 | 2007-11-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2010-11-01 | 2011-05-03 | 2011-11-02 | $7700.00 | Window Closed / Lapsed |