Method For Detecting a Target Nucleic Acid in a Sample
First Claim
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1. A method for detecting a target nucleic acid in a sample comprising:
- a) contacting said sample with a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity and a first and a second oligonucleotide probe, under conditions wherein said first and second oligonucleotide probes selectively hybridize to said target nucleic acid, wherein the first oligonucleotide probe includes a fluorescent reporter and the second oligonucleotide probe includes a quencher so that the fluorescence of the fluorescent reporter is quenched by the quencher when the first and second oligonucleotide probes are hybridized to the target nucleic acid and the fluorescence of the fluorescent reporter is unquenched by the quencher when the first and second oligonucleotide are not hybridized to the target nucleic acid,b) exciting the fluorescent reporter with a light source,c) monitoring the fluorescence of the fluorescent reporter under conditions where the first and second oligonudeotide probes are hybridized to the target nucleic acid, andd) comparing the fluorescence of step c) to that obtained under conditions where the first and second oligonucleotide probes are not hybridized to the target nucleic acid.
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Abstract
The invention relates to a method for detecting a target nucleic acid in a sample using fluorescent probe pairs which include fluorescent reporter and quencher molecules which may be used in hybridization assays and in nucleic acid amplification reactions, especially polymerase chain reactions (PCR).
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Citations
16 Claims
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1. A method for detecting a target nucleic acid in a sample comprising:
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a) contacting said sample with a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity and a first and a second oligonucleotide probe, under conditions wherein said first and second oligonucleotide probes selectively hybridize to said target nucleic acid, wherein the first oligonucleotide probe includes a fluorescent reporter and the second oligonucleotide probe includes a quencher so that the fluorescence of the fluorescent reporter is quenched by the quencher when the first and second oligonucleotide probes are hybridized to the target nucleic acid and the fluorescence of the fluorescent reporter is unquenched by the quencher when the first and second oligonucleotide are not hybridized to the target nucleic acid,b) exciting the fluorescent reporter with a light source, c) monitoring the fluorescence of the fluorescent reporter under conditions where the first and second oligonudeotide probes are hybridized to the target nucleic acid, and d) comparing the fluorescence of step c) to that obtained under conditions where the first and second oligonucleotide probes are not hybridized to the target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A kit for detecting a target nucleic acid in a sample comprising:
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a first oligonucleotide probe comprising a fluorescent reporter, a second oligonucleotide probe comprising a quencher effective to quench the fluorescence of the fluorescent reporter when the first and second oligonucleotide probes are hybridized to the target nucleic acid, and a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity. - View Dependent Claims (8, 9, 10, 11)
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12. A reaction mixture comprising:
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a target nucleic acid in a sample, a nucleic acid polymerase substantially lacking 5′
-3′
nuclease activity,a first oligonucleotide probe comprising a fluorescent reporter, and a second oligonucleotide probe comprising a quencher effective to quench the fluorescence of the fluorescent reporter when the first and second oligonucleotide probes are hybridized to the target nucleic acid. - View Dependent Claims (13, 14, 15, 16)
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Specification