Enhanced nucleic acid identification and detection

Abstract

The present invention relates to assays, including amplification assays, conducted in the presence of modulators. These assays can be used to detect the presence of particular nucleic acid sequences. In particular, these assays can allow for genotyping or other genetic analysis.

1 .- 56 . (canceled) 
     
     
         57 . A method, comprising:
 providing a volume suspected of containing a target nucleic acid;   conducting an isothermal nucleic acid amplification reaction in said volume in presence of a modulator comprising an engineered or non-natural sequence-specific nuclease,   
       wherein said modulator modulates said isothermal nucleic acid amplification in the presence of said target nucleic acid, and 
       wherein said modulator acts on said target nucleic acid within a region amplified by said isothermal nucleic acid amplification reaction. 
     
     
         58 . The method of  claim 57 , further comprising
 after the providing and prior to the conducting, dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals,   wherein the conducting is performed in the dispersed volume in said plurality of areas.   
     
     
         59 . The method of  claim 58 , wherein said plurality of areas each comprises at most one copy of said target nucleic acid. 
     
     
         60 . The method of  claim 58 , further comprising prior to the conducting, providing the modulator to said plurality of areas. 
     
     
         61 . The method of  claim 57 , wherein the providing comprises providing a first volume comprising a first nucleic acid and a second volume comprising a second nucleic acid. 
     
     
         62 . The method of  claim 61 , further comprising
 after the providing and prior to the conducting, dispersing said first volume among a plurality of first areas and dispersing said second volume among a plurality of second areas,   wherein the conducting is performed in the dispersed first volume in said plurality of first areas and in the dispersed second volume in said plurality of second areas.   
     
     
         63 . The method of  claim 62 , wherein said plurality of first areas each comprise at most one copy of said first nucleic acid and said plurality of second areas each comprise at most one copy of said second nucleic acid. 
     
     
         64 . The method of  claim 62 , further comprising prior to the conducting, providing the modulator to said plurality of first areas or to said plurality of second areas. 
     
     
         65 . The method of  claim 61 , further comprising detecting a difference in said isothermal nucleic acid amplification reaction between said first nucleic acid and said second nucleic acid. 
     
     
         66 . The method of  claim 65 , wherein said difference is diagnostic of the presence of the target nucleic acid within said first nucleic acid and said second nucleic acid. 
     
     
         67 . The method of  claim 57 , wherein said modulation comprises producing a difference in amplification efficiency. 
     
     
         68 . The method of  claim 67 , wherein the method further comprises
 after the providing and prior to the conducting, dispersing said volume among a plurality of areas, such that said plurality of areas comprises a distribution of nucleic acids said distribution capable of generating digital nucleic acid amplification signals,   wherein the conducting is performed in the dispersed volume in said plurality of areas; and   
       wherein said difference in amplification efficiency produces a positive amplification signal in a subset of said plurality of areas. 
     
     
         69 . The method of  claim 68 , wherein said positive amplification signal in said subset of said plurality of areas is diagnostic of the presence of said target nucleic acid within said volume. 
     
     
         70 . The method of  claim 57 , wherein the target nucleic acid comprises an HCV nucleic acid. 
     
     
         71 . The method of  claim 70 , wherein said method generates a signal from which an HCV genotype can be determined. 
     
     
         72 . The method of  claim 57 , further comprising comparing results of said isothermal nucleic acid amplification reaction to results of a control isothermal nucleic acid amplification reaction carried out in the absence of the modulator. 
     
     
         73 . The method of  claim 57 , wherein said isothermal nucleic acid amplification reaction is selected from the group consisting of Recombinase Polymerase Amplification (RPA), Loop-mediated isothermal amplification (LAMP), Helicase-dependent amplification (HAD), Strand displacement amplification (SDA), Nucleic acid sequence based amplification (NASBA), and Nicking enzyme amplification reaction (NEAR). 
     
     
         74 . The method of  claim 73 , wherein said isothermal nucleic acid amplification reaction is LAMP or NASBA. 
     
     
         75 . The method of  claim 57 , wherein the nuclease is selected from the group consisting of zinc-finger nuclease, transcription activator-like effector nuclease, meganuclease and RNA-guided Cas9 nuclease. 
     
     
         76 . The method of  claim 57 , wherein the modulator is RNA-guided Cas9 nuclease. 
     
     
         77 . The method of  claim 76 , wherein the modulator further comprises a crRNA and a tracrRNA, the crRNA, tracRNA and Cas9 forming a Cas9-crRNA-tracrRNA complex.