Methods of detecting a target nucleic acid
The invention relates to compositions and methods of generating a signal indicative of the presence of a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe. The invention also includes the steps of cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid in a sample.
1 - 48 . (canceled)
49 . A method of detecting a nucleic acid molecule, comprising: a) exposing a sample comprising a target nucleic acid molecule to a purified thermostable FEN-1 endonuclease to generate a cleavage product; and b) detecting said cleavage product.
50 . The method of claim 49 , wherein said cleavage product is produced from a probe oligonucleotide hybridized to said target nucleic acid.
51 . The method of claim 49 , wherein step a) comprises: exposing said sample to said purified thermostable FEN-1 endonuclease and a polymerase.
52 . The method of claim 51 , wherein said polymerase comprises a template-dependent polymerase.
53 . The method of claim 49 , wherein said detecting comprises detection of fluorescence.
54 . The method of claim 49 , wherein said target nucleic acid comprises synthetic target nucleic acid.
55 . The method of claim 54 , wherein said synthetic target nucleic comprises an amplified nucleic acid.
56 . The method of claim 55 , wherein said amplified nucleic acid is produced using a polymerase chain reaction.
57 . The method of claim 49 , wherein said method is conducted under isothermal conditions.
58 . The method of claim 49 , wherein said exposing comprises hybridizing first and second nucleic acid molecules to said target nucleic acid.
59 . The method of claim 49 , wherein said detecting comprises detecting a non-target cleavage product.
60 . The method of claim 49 , wherein said thermostable FEN-1 endonuclease comprises a FEN-1 endonuclease from an archaebacterial species.
61 . The method of claim 60 , wherein said archaeal FEN-1 endonuclease comprises a FEN-1 endonuclease from Pyrococcus furiosus.