Invasive cleavage of nucleic acids
First Claim
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1. A method for detecting the presence of a target nucleic acid molecule in a sample, comprising:
- a) incubating a sample with a thermostable 5′
nuclease under conditions wherein a cleavage structure is formed, said cleavage structure comprising;
i) a synthetic target nucleic acid, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;
ii) a first nucleic acid molecule, wherein at least a portion of said first nucleic acid molecule is completely complementary to said first region of said target nucleic acid;
iii) a second nucleic acid molecule comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second region of said target nucleic acid;
wherein said thermostable 5′
nuclease lacks synthesis activity, and wherein at least a portion of said first nucleic acid molecule is annealed to said first region of said synthetic target nucleic acid, and wherein at least a portion of said second nucleic acid molecule is annealed to said second region of said synthetic target nucleic acid;
b) cleaving said cleavage structure with said thermostable 5′
nuclease so as to generate non-target cleavage product; and
c) detecting the cleavage of said cleavage structure.
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Abstract
The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.
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Citations
57 Claims
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1. A method for detecting the presence of a target nucleic acid molecule in a sample, comprising:
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a) incubating a sample with a thermostable 5′
nuclease under conditions wherein a cleavage structure is formed, said cleavage structure comprising;
i) a synthetic target nucleic acid, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;
ii) a first nucleic acid molecule, wherein at least a portion of said first nucleic acid molecule is completely complementary to said first region of said target nucleic acid;
iii) a second nucleic acid molecule comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second region of said target nucleic acid;
wherein said thermostable 5′
nuclease lacks synthesis activity, and wherein at least a portion of said first nucleic acid molecule is annealed to said first region of said synthetic target nucleic acid, and wherein at least a portion of said second nucleic acid molecule is annealed to said second region of said synthetic target nucleic acid;
b) cleaving said cleavage structure with said thermostable 5′
nuclease so as to generate non-target cleavage product; and
c) detecting the cleavage of said cleavage structure. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A method for detecting the presence of a target nucleic acid molecule in a sample, comprising:
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a) incubating a sample with a thermostable FEN-1 nuclease under conditions wherein a cleavage structure is formed, said cleavage structure comprising;
i) a synthetic target nucleic acid, said synthetic target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;
ii) a first nucleic acid molecule, wherein at least a portion of said first nucleic acid molecule is completely complementary to said first region of said target nucleic acid;
iii) a second nucleic acid molecule comprising a 3′
portion and a 5′
portion, wherein said 5′
portion is completely complementary to said second region of said target nucleic acid;
wherein at least a portion of said first nucleic acid molecule is annealed to said first region of said synthetic target nucleic acid, and wherein at least a portion of said second nucleic acid molecule is annealed to said second region of said synthetic target nucleic acid;
b) cleaving said cleavage structure with said thermostable FEN-1 nuclease so as to generate non-target cleavage product; and
c) detecting the cleavage of said cleavage structure. - View Dependent Claims (32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)
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Specification