Simultaneous sequencing of nucleic acids
First Claim
1. 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.
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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.
165 Citations
23 Claims
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1. A method for the simultaneous and specific labeling of nucleic acids comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for the simultaneous sequencing of nucleic acids comprising:
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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. - View Dependent Claims (22, 23)
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Specification