Methods for detection of a target nucleic acid by forming a cleavage structure using an RNA polymerase
First Claim
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1. A composition for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, the composition comprising an RNA polymerase, a FEN nuclease, and a probe that is complementary to the target nucleic acid.
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Abstract
The invention relates to compositions and methods for generating a signal indicative of the presence of a target nucleic acid in a sample, where the compositions and methods include an RNA polymerase, a FEN nuclease, and a probe.
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Citations
47 Claims
- 1. A composition for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, the composition comprising an RNA polymerase, a FEN nuclease, and a probe that is complementary to the target nucleic acid.
- 15. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, the composition comprising an RNA polymerase, a FEN nuclease, and a probe that is complementary to the target nucleic acid.
- 26. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a cleavage structure by incubating a sample comprising a target nucleic acid sequence with an RNA polymerase and cleaving the cleavage structure with a FEN nuclease to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid sequence in the sample.
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27. A method of detecting or measuring a target nucleic acid sequence, comprising forming a cleavage structure by incubating a sample comprising a target nucleic acid sequence with an RNA polymerase, cleaving the cleavage structure with a FEN nuclease to release a nucleic acid fragment and detecting and/or measuring the release of the fragment as an indication of the presence of the target sequence in the sample.
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36. A method for detecting a target nucleic acid sequence in a sample, comprising:
- mixing a probe, a target nucleic acid having a promoter region, a FEN nuclease and an RNA polymerase, under conditions which are permissive for the steps of (i) binding of the RNA polymerase to the promoter region in the target nucleic acid sequence and annealing of the probe to the target, (ii) synthesizing an RNA polymerase extension product, (iii) forming a cleavage structure, and (iv) and cleaving the cleavage structure with the FEN nuclease and detecting and/or measuring the release of labeled fragments or cleavage of the probe as an indication of the presence of the target sequence in the sample.
- View Dependent Claims (38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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37. A method for detecting a target nucleic acid sequence in a sample, comprising:
- mixing a probe, a target nucleic acid, a FEN nuclease, a primer and an RNA polymerase, under conditions which are permissive for the steps of (i) annealing of the primer and probe to the target nucleic acid, (ii) synthesizing an RNA polymerase extension product from said primer, (iii) forming a cleavage structure, and (iv) and cleaving the cleavage structure with the FEN nuclease and detecting and/or measuring the release of labeled fragments or cleavage of the probe as an indication of the presence of the target sequence in the sample.
Specification