Detection of target nucleic acid molecules using thermostable 5' nuclease
First Claim
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1. A method of detecting the presence of a nucleic acid molecule comprising:
- a) providing;
i) a thermostable 5'"'"' nuclease derived from a thermostable polymerase modified to have reduced synthetic activity, wherein said 5'"'"' nuclease is capable of cleaving a linear nucleic acid duplex structure so as to create substantially a single, single-stranded nucleic acid cleavage product,ii) a target nucleic acid having a first and a second portion,iii) a first oligonucleotide complementary to said first portion of said target nucleic acid, andiv) a second oligonucleotide having a 5'"'"' and a 3'"'"' end, said second oligonucleotide comprising a 5'"'"' region and a 3'"'"' region, wherein said 3'"'"' region is complementary to said second portion of said target nucleic acid and said 5'"'"' region is non-complementary to said target nucleic acid and provides a single-stranded arm;
b) mixing said 5'"'"' nuclease, said target nucleic acid, said first oligonucleotide and said second oligonucleotide under conditions wherein said first oligonucleotide and said 3'"'"' region of said second oligonucleotide are annealed to said target nucleic acid sequence so as to create a first cleavage structure and cleavage of said first cleavage structure results in the liberating of said single-stranded arm of said second oligonucleotide to generate a first signal oligonucleotide and a cleaved second oligonucleotide; and
c) detecting the presence of said first signal oligonucleotide.
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Abstract
A means for cleaving a nucleic acid cleavage structure in a site-specific manner is disclosed. A cleaving enzyme having 5'"'"' nuclease activity without interfering nucleic acid synthetic ability is employed as the basis of a novel method of detection of specific nucleic acid sequences.
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Citations
20 Claims
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1. A method of detecting the presence of a nucleic acid molecule comprising:
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a) providing; i) a thermostable 5'"'"' nuclease derived from a thermostable polymerase modified to have reduced synthetic activity, wherein said 5'"'"' nuclease is capable of cleaving a linear nucleic acid duplex structure so as to create substantially a single, single-stranded nucleic acid cleavage product, ii) a target nucleic acid having a first and a second portion, iii) a first oligonucleotide complementary to said first portion of said target nucleic acid, and iv) a second oligonucleotide having a 5'"'"' and a 3'"'"' end, said second oligonucleotide comprising a 5'"'"' region and a 3'"'"' region, wherein said 3'"'"' region is complementary to said second portion of said target nucleic acid and said 5'"'"' region is non-complementary to said target nucleic acid and provides a single-stranded arm; b) mixing said 5'"'"' nuclease, said target nucleic acid, said first oligonucleotide and said second oligonucleotide under conditions wherein said first oligonucleotide and said 3'"'"' region of said second oligonucleotide are annealed to said target nucleic acid sequence so as to create a first cleavage structure and cleavage of said first cleavage structure results in the liberating of said single-stranded arm of said second oligonucleotide to generate a first signal oligonucleotide and a cleaved second oligonucleotide; and c) detecting the presence of said first signal oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method of detecting the presence of a nucleic acid molecule comprising:
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a) providing; i) a thermostable 5'"'"' nuclease derived from a thermostable polymerase modified to have reduced synthetic activity, ii) a target nucleic acid, iii) an oligonucleotide complementary to a portion of said target nucleic acid, said oligonucleotide being present in excess relative to said target nucleic acid; b) mixing said thermostable 5'"'"' nuclease, said target nucleic acid and said oligonucleotide under conditions wherein a first molecule of said oligonucleotide anneals to said target nucleic acid so as to create a duplex nucleic acid substrate thereby permitting the digestion of said oligonucleotide and the release of said digested oligonucleotide from said target nucleic acid structure. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification