USE OF THERMOSTABLE ENDONUCLEASES FOR GENERATING REPORTER MOLECULES
First Claim
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1. A method for amplifying a target nucleic acid, or portion thereof, in a nucleic acid composition, which comprises:
- (a) contacting, under hybridization conditions, a nucleic acid composition with an oligonucleotide and a primer polynucleotide, wherein the oligonucleotide comprises;
(i) a nucleotide subsequence complementary to the target nucleic acid, and(ii) a non-terminal and non-functional portion of a first endonuclease cleavage site; and
(b) extending the oligonucleotide under amplification conditions, thereby generating an extended oligonucleotide, wherein the primer polynucleotide hybridizes to the extended oligonucleotide and is extended under the amplification conditions, thereby yielding a double-stranded amplification product that comprises a functional first endonuclease cleavage site, whereby the target nucleic acid, or portion thereof, is amplified.
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Abstract
Provided are compositions and methods for amplifying, capturing and/or detecting target nucleic acids using cleavable oligonucleotides.
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Citations
32 Claims
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1. A method for amplifying a target nucleic acid, or portion thereof, in a nucleic acid composition, which comprises:
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(a) contacting, under hybridization conditions, a nucleic acid composition with an oligonucleotide and a primer polynucleotide, wherein the oligonucleotide comprises; (i) a nucleotide subsequence complementary to the target nucleic acid, and (ii) a non-terminal and non-functional portion of a first endonuclease cleavage site; and (b) extending the oligonucleotide under amplification conditions, thereby generating an extended oligonucleotide, wherein the primer polynucleotide hybridizes to the extended oligonucleotide and is extended under the amplification conditions, thereby yielding a double-stranded amplification product that comprises a functional first endonuclease cleavage site, whereby the target nucleic acid, or portion thereof, is amplified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for detecting the presence or absence of a target nucleic acid in a nucleic acid composition, which comprises:
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(a) contacting, under hybridization conditions, a nucleic acid composition with an oligonucleotide and a primer polynucleotide, wherein the oligonucleotide comprises; (i) a nucleotide subsequence complementary to the target nucleic acid, (ii) a non-terminal and non-functional portion of a first endonuclease cleavage site, and (iii) a detectable feature; and (b) exposing the nucleic acid composition to amplification conditions, wherein (i) the oligonucleotide is extended when the target nucleic acid is present, and (ii) the primer polynucleotide hybridizes to the extended oligonucleotide and is extended under the amplification conditions, thereby yielding a double-stranded amplification product that comprises a functional first endonuclease cleavage site; (c) contacting the nucleic acid composition with a first endonuclease that cleaves the functional first endonuclease cleavage site, thereby generating a cleavage product comprising the detectable feature; and (d) detecting the presence or absence of the cleavage product comprising the detectable feature, whereby the presence or absence of the target nucleic acid is detected based on the presence or absence of the cleavage product comprising the detectable feature. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification