Cleavage of nucleic acids
First Claim
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1. A method for detecting the presence of a target nucleic acid molecule comprising:
- a) forming a cleavage structure between a target nucleic acid, a first oligonucleotide, and a second oligonucleotide, wherein the target nucleic acid comprises a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region, wherein said first oligonucleotide comprises a 3′
portion and a 5′
portion, wherein said 3′
portion of said first oligonucleotide contains a sequence of one or more nucleotides that is completely complementary to said third region of said target nucleic acid, and wherein said 5′
portion of said first oligonucleotide contains a sequence of one or more nucleotides contiguous to said 3′
portion of said first oligonucleotide that is completely complementary to said second region of said target nucleic acid, wherein said second oligonucleotide comprises a 3′
portion and a 5′
portion, wherein said 5′
portion contains a sequence or one or more nucleotides that are completely complementary to said first region of said target nucleic acid, wherein said cleavage structure is formed when said first oligonucleotide is annealed to said third and said second region of said target nucleic acid and wherein said second oligonucleotide is annealed to said first region of said target nucleic acid such that the 3′
portion of said second oligonucleotide overlaps with the sequence of nucleotides in said 5′
portion of said first oligonucleotide annealed to said second region of said target nucleic acid;
b) cleaving said cleavage structure with a thermostable enzyme having 5′
nuclease activity and substantially lacking nucleic acid polymerase activity to generate a non-target cleavage product, andc) detecting the cleavage of said cleavage structure to detect the presence of the target nucleic acid molecule.
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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
27 Claims
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1. A method for detecting the presence of a target nucleic acid molecule comprising:
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a) forming a cleavage structure between a target nucleic acid, a first oligonucleotide, and a second oligonucleotide, wherein the target nucleic acid comprises a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region, wherein said first oligonucleotide comprises a 3′
portion and a 5′
portion, wherein said 3′
portion of said first oligonucleotide contains a sequence of one or more nucleotides that is completely complementary to said third region of said target nucleic acid, and wherein said 5′
portion of said first oligonucleotide contains a sequence of one or more nucleotides contiguous to said 3′
portion of said first oligonucleotide that is completely complementary to said second region of said target nucleic acid, wherein said second oligonucleotide comprises a 3′
portion and a 5′
portion, wherein said 5′
portion contains a sequence or one or more nucleotides that are completely complementary to said first region of said target nucleic acid, wherein said cleavage structure is formed when said first oligonucleotide is annealed to said third and said second region of said target nucleic acid and wherein said second oligonucleotide is annealed to said first region of said target nucleic acid such that the 3′
portion of said second oligonucleotide overlaps with the sequence of nucleotides in said 5′
portion of said first oligonucleotide annealed to said second region of said target nucleic acid;b) cleaving said cleavage structure with a thermostable enzyme having 5′
nuclease activity and substantially lacking nucleic acid polymerase activity to generate a non-target cleavage product, andc) detecting the cleavage of said cleavage structure to detect the presence of the target nucleic acid molecule. - 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)
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Specification