Methods for detection of 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 detecting a target nucleic acid comprising exposing a composition comprising a purified thermostable FEN-1 endonuclease and a polymerase 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 a FEN-1 endonuclease from an archaebacterial species.  
     
     
         51 . The method of  claim 50 , wherein said archaeal FEN-1 endonuclease comprises a FEN-1 endonuclease from  Pyrococcus furiosus.    
     
     
         52 . The method of  claim 49 , wherein said polymerase comprises a thermostable polymerase.  
     
     
         53 . The method of  claim 49 , wherein said polymerase comprises a template-dependent polymerase.  
     
     
         54 . The method of  claim 49 , wherein said purified thermostable FEN-1 endonuclease and said polymerase are in a mixture.  
     
     
         55 . The method of  claim 49 , wherein said mixture comprises a reaction mixture.  
     
     
         56 . The method of  claim 49 , wherein said exposing comprises hybridizing a nucleic acid molecule to said target nucleic acid.  
     
     
         57 . The method of  claim 56 , wherein said nucleic acid molecule comprises a probe oligonucleotide.  
     
     
         58 . The method of  claim 56 , wherein said nucleic acid molecule is attached to a solid support.  
     
     
         59 . The method of  claim 56 , wherein said nucleic acid molecule comprises a label.  
     
     
         60 . The method of  claim 59 , wherein said label comprises a fluorescent label.  
     
     
         61 . The method of  claim 49 , wherein said exposing comprises hybridizing first and second nucleic acid molecules 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 66 , 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.