Patent ID: 7482118

Claim:
A method for detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, comprising: a) providing: a Pfu FEN-1 nuclease comprising amino acid sequence SEQ ID NO:79 with the exception of amino acid position 94, wherein said Pfu FEN-1 comprises a Y33 residue and a mutation in the R94 residue, wherein the mutation in said R94 residue is R94A; a source of target nucleic acid, said target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region; a first oligonucleotide, wherein a first portion of said first oligonucleotide comprises at least one nucleotide analog and wherein said first portion is completely complementary to said first portion of said first target nucleic acid, and; an aromatic moiety; b) combining said Pfu FEN-1 nuclease, said target nucleic acid, said first oligonucleotide, and said aromatic moiety under reaction conditions to form a cleavage complex, wherein said first portion of said first oligonucleotide is annealed to said first region of said target nucleic acid to form a duplex, wherein said Pfu FEN-1 nuclease recognizes said duplex, and wherein said aromatic moiety interacts with said Y33 residue of said Pfu FEN-1 nuclease, such that cleavage of said complex occurs to generate a non-target cleavage product; and c) detecting the cleavage of said cleavage complex.