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 characterizing a nucleic acid molecule comprising: a) exposing a sample comprising a nucleic acid molecule to a composition comprising an isolated thermostable enzyme having 5′ nuclease activity but substantially lacking polymerase activity and an isolated polymerase under conditions such that said nucleic acid molecule is altered; and b) detecting the alteration of said nucleic acid molecule.
50 . The method of claim 49 , wherein said alteration comprises cleavage of said nucleic acid molecule.
51 . The method of claim 50 , wherein said cleavage occurs by said thermostable enzyme having 5′ nuclease activity after said nucleic acid molecule is reacted with said polymerase.
52 . The method of claim 50 , wherein said nucleic acid molecule is hybridized to a target nucleic acid molecule.
53 . The method of claim 49 , wherein said exposing occurs under conditions permissive for primer extension by a polymerase.
54 . The method of claim 53 , wherein said conditions permit exponential amplification of a target nucleic acid molecule.
55 . The method of claim 52 , wherein said target nucleic acid molecule is DNA.
56 . The method of claim 52 , wherein said target nucleic acid molecule is RNA.
57 . The method of claim 52 , wherein said target nucleic acid is cDNA.
58 . The method of claim 48 , wherein said target nucleic acid molecule contains a nucleotide analogue.
59 . The method of claim 52 , wherein said target nucleic acid molecule is from a pathogen.
60 . The method of claim 52 , wherein said target nucleic acid is synthetic.
61 . The method of claim 60 , wherein said target nucleic acid is produced by amplification.
62 . The method of claim 61 , wherein said amplification comprises a polymerase chain reaction.
63 . The method of claim 60 , wherein said pathogen is a virus.
64 . The method of claim 60 , wherein said pathogen is a bacterium.
65 . The method of claim 49 , wherein said sample is serum.
66 . The method of claim 49 , wherein said sample is tissue.
67 . The method of claim 49 , wherein said nucleic acid molecule is attached to a solid support.
68 . The method of claim 49 , wherein said nucleic acid molecule comprises a label.
69 . The method of claim 68 , wherein said label comprises a fluorescent label.