Methods of detecting a target nucleic acid

Abstract

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.