Cleavage of nucleic acids
DCFirst Claim
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1. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
- a) providing;
i) a cleavage means,ii) a source of target nucleic acid, said target nucleic acid having a first region, a second region, a third region and a fourth region, wherein said first region is located downstream from said second region, said second region is contiguous to and downstream from said third region and said third region is located adjacent to and downstream from said fourth region;
iii) a first oligonucleotide complementary to said fourth region of said target nucleic acid;
iv) second and third oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said second oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said second oligonucleotide and said 3'"'"' portion of said third oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5'"'"' portion of said third oligonucleotide contains sequence complementary to said first region of said target nucleic acid;
b) mixing said cleavage means, said target nucleic acid, said first oligonucleotide, said second oligonucleotide and said third oligonucleotide to create a reaction mixture under reaction conditions such that said first oligonucleotide is annealed to said fourth region of said target nucleic acid and wherein at least said 3'"'"' portion of said second oligonucleotide is annealed to said target nucleic acid and wherein at least said 5'"'"' portion of said third oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products, each non-target cleavage product having a 3'"'"'-hydroxyl group; and
c) detecting said non-target cleavage products.
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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 of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
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a) providing; i) a cleavage means, ii) a source of target nucleic acid, said target nucleic acid having a first region, a second region, a third region and a fourth region, wherein said first region is located downstream from said second region, said second region is contiguous to and downstream from said third region and said third region is located adjacent to and downstream from said fourth region; iii) a first oligonucleotide complementary to said fourth region of said target nucleic acid; iv) second and third oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said second oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said second oligonucleotide and said 3'"'"' portion of said third oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5'"'"' portion of said third oligonucleotide contains sequence complementary to said first region of said target nucleic acid; b) mixing said cleavage means, said target nucleic acid, said first oligonucleotide, said second oligonucleotide and said third oligonucleotide to create a reaction mixture under reaction conditions such that said first oligonucleotide is annealed to said fourth region of said target nucleic acid and wherein at least said 3'"'"' portion of said second oligonucleotide is annealed to said target nucleic acid and wherein at least said 5'"'"' portion of said third oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products, each non-target cleavage product having a 3'"'"'-hydroxyl group; and c) detecting said non-target cleavage products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
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a) providing; i) a cleavage means, ii) a source of target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region; iii) first and second oligonucleotides having 3'"'"' and 5'"'"' portions, wherein said 3'"'"' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein said 5'"'"' portion of said second oligonucleotide contains sequence complementary to said first region of said target nucleic acid; b) mixing, in any order, said cleavage means, said target nucleic acid, said first oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3'"'"' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5'"'"' portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products, each non-target cleavage product having a 3'"'"'-hydroxyl group; and c) detecting said non-target cleavage products. - View Dependent Claims (17, 18, 19, 20, 21)
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22. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products comprising:
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a) providing; i) a cleavage means, ii) a source of target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is downstream from said second region and wherein said second region is contiguous to and downstream from said third region; iii) a first and second oligonucleotide having 3'"'"' and 5'"'"' portion, wherein said 3'"'"' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5'"'"' portion of said first oligonucleotide and said 3'"'"' portion of said second oligonucleotied each contain sequence fully complementary to said second region of said target nucleic acid, and wherein 5'"'"' portion of said second oliginucleotide contains sequence complementary to said first region of said target nucleic acid; b) mixing, in any order, said cleavage means, said target nucleic acid, said first oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3'"'"' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5'"'"' portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products, each non-target cleavage product having a 3'"'"'-hydroxyl group; and c) detecting said non-target cleavage products. - View Dependent Claims (23, 24, 25, 26, 27)
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Specification