Invasive cleavage of nucleic acids.

摘要

A method for detecting the presence of a nucleic acid target 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, second region and a third region, said first region downstream of and contiguous to said second region and said second region being downstream of and contiguous to said third region, wherein said first oligonucleotide comprises a 3 '' and 5 '', containing said 3 '' of said first oligonucleotide a sequence of one or more nucleotides that is completely complementary to said third region of said target nucleic acid, and containing said 5 'of said first oligonucleotide sequence one or more adjacent nucleotide said 3 '' of said first oligonucleotide that is completely complementary to said segund a region of said target nucleic acid, wherein said second oligonucleotide comprises a 3 '' and 5 '', containing said 5 'sequence of one or more nucleotides that is completely complementary to said first region of said target nucleic acid and wherein said 3 '' of said second oligonucleotide is complementary to said second region of said target nucleic acid or comprises or consists of at least one nucleotide is wrongly matched to said second region of said acid target nucleic, said structure formed cleavage when said first oligonucleotide to said third and said second region of said target nucleic acid anneals, and wherein said second oligonucleotide is annealed to said first region of said target nucleic acid such that the part 3 '' of said second oligonucleotide overlaps with the nucleotide sequence of said 5 'of said first oligonucleotide mated to said second region of said target nucleic acid; b) cleaving said cleavage structure with a thermostable enzyme having 5 'nuclease activity "and while lacking synthetic activity or comprising reduced synthetic activity compared to a native DNA polymerase to generate a cleavage product non-target, and c) detecting cleavage of said cleavage structure to detect the presence of the target nucleic acid molecule.