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 for detecting a target nucleic acid comprising: exposing a composition comprising a purified thermostable FEN-1 endonuclease and a purified nucleic acid molecule to a sample suspected of containing a target nucleic under conditions such that said target nucleic acid, if present in said sample, is detected.
50 . The method of claim 49 , wherein said thermostable FEN-1 endonuclease comprises an FEN-1 endonuclease from an archaebacterial species.
51 . The method of claim 50 , wherein said FEN-1 endonuclease comprises a FEN-1 endonuclease from Pyrococcus furiosus.
52 . The method of claim 49 , further comprising a polymerase.
53 . The method of claim 52 , wherein said polymerase comprises a thermostable polymerase.
54 . The method of claim 52 , wherein said polymerase comprises a template-dependent polymerase.
55 . The method of claim 49 , wherein said purified thermostable FEN-1 endonuclease and said purified nucleic acid molecule are in a mixture.
56 . The method of claim 55 , wherein said mixture comprises a reaction mixture.
57 . The method of claim 49 , wherein said exposing comprises hybridizing a second nucleic acid molecule to said target nucleic acid.
58 . The method of claim 57 , wherein said second nucleic acid molecule comprises a probe oligonucleotide.
59 . The method of claim 57 , wherein said second nucleic acid molecule comprises a label.
60 . The method of claim 59 , wherein said label comprises a fluorescent label.
61 . The method of claim 57 , wherein said exposing comprises hybridizing said purified nucleic acid molecule and said second nucleic acid molecule to said target nucleic acid.
62 . The method of claim 49 , wherein said detecting comprises detecting a non-target cleavage product.
63 . The method of claim 49 , wherein said detecting the cleavage of said cleavage structure comprises detection selected from the group consisting of detection of radioactivity, luminescence, phosphorescence, and fluorescence polarization.
64 . The method of claim 49 , wherein said detecting the cleavage of said cleavage structure comprises detection based on the charge of non-target cleavage product.
65 . The method of claim 49 , wherein said target nucleic acid is DNA.
66 . The method of claim 49 , wherein said target nucleic acid is synthetic nucleic acid.
67 . The method of claim 66 , wherein said synthetic nucleic acid comprises amplified nucleic acid.
68 . The method of claim 67 , wherein said synthetic nucleic acid is generated by a polymerase chain reaction.
69 . The method of claim 49 , wherein said target nucleic acid is RNA.