Patent ID: 7011944

Claim:
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.