Detection of nucleic acid sequences by invader-directed cleavage

A method of detecting the presence of a target RNA molecule by detecting non-target cleavage products comprising:

• a) providing:
• i) a cleavage means,
• ii) a source of target RNA, said target RNA 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 full 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 RNA, 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 RNA and wherein at least said 5' portion of said second oligonucleotide is annealed to said target RNA so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5' and 3' portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 3' portion of said first oligonucleotide and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products;
• and
• c) detecting said non-target cleavage products.