Real Time Cleavage Assay

Abstract

A cleavage-based real-time PCR assay method is provided. In general terms, the assay method includes subjecting a reaction mixture comprising a) PCR reagents for amplifying a nucleic acid target, and b) flap cleavage reagents for performing a flap cleavage assay on the amplified nucleic acid target to two sets of thermocycling conditions. No additional reagents are added to the reaction between said first and second sets of cycles and, in each cycle of the second set of cycles, cleavage of a flap probe is measured.

1 - 13 . (canceled) 
     
     
         14 . A method of sample analysis comprising:
 (a) subjecting a reaction mixture comprising:
 a target nucleic acid; 
 PCR reagents for amplifying the target nucleic acid, and 
 flap cleavage reagents for performing a flap cleavage assay on the target nucleic acid, 
   to thermocycling conditions that include multiple cycles of:
 a first temperature of at least 90° C.; 
 a second temperature; and 
 a third temperature in the range of 65° C. to 75° C.; 
 wherein the second temperature is at least 10° C. lower than the third temperature; and 
   (b) measuring cleavage of a flap oligonucleotide while the reaction mixture is at the second temperature.   
     
     
         15 . The method of  claim 14 , wherein said second temperature is in the range 50° C. to 55° C. 
     
     
         16 . The method of  claim 14 , wherein the measuring step (b) comprises measuring cleavage of a 5′ fluorophore from the flap oligonucleotide. 
     
     
         17 . The method of  claim 14 , wherein the multiple cycles is 20-50 cycles. 
     
     
         18 . The method of  claim 17 , wherein said method further comprises graphing the amount of cleavage that occurs in each of the 20-50 cycles, thereby providing an estimate of the abundance of the target nucleic acid. 
     
     
         19 . The method of  claim 14 , wherein the reaction mixture has a volume that is in the range of 5 μl to 200 μl, and each of said first to third temperatures is, independently, of a duration in the range of 10 seconds to 3 minutes. 
     
     
         20 . The method of  claim 14 , wherein said cleavage of the flap oligonucleotide in (b) indicates a mutation in the target nucleic acid. 
     
     
         21 . The method of  claim 14 , wherein:
 the PCR reagents comprise: a nucleic acid template; a first PCR primer, a second PCR primer, a thermostable polymerase, and nucleotides; and   the flap cleavage reagents comprise: an invasive oligonucleotide, a flap oligonucleotide, a thermostable flap endonuclease and a FRET cassette,   wherein i. the first PCR primer and the invasive oligonucleotide have different nucleotide sequences or ii. the first PCR primer and the invasive oligonucleotide have the same nucleotide sequence and the first primer is used as the invasive oligonucleotide   
     
     
         22 . The method of  claim 21 , wherein said first PCR primer is present in said reaction mixture at the same concentration as the second PCR primer. 
     
     
         23 . The method of  claim 21 , wherein the thermostable polymerase is selected from Taq, Pfu, Pwo, UlTma and Vent. 
     
     
         24 . The method of  claim 14 , wherein the target nucleic acid corresponds to a mutated locus in the human genome. 
     
     
         25 . The method of  claim 24 , wherein the mutated locus is cancer-related. 
     
     
         26 . The method of  claim 25 , wherein the mutated locus is an oncogenic mutation in PIK3CA, NRAS, KRAS, JAK2, HRAS, FGFR3, FGFR1, EGFR, CDK4, BRAF, RET, PGDFRA, KIT or ERBB2. 
     
     
         27 . The method of  claim 14 , wherein no additional reagents are added to said reaction mix between steps (a) and (b). 
     
     
         28 . The method of  claim 14 , wherein said reaction mix further comprises PCR reagents and flap reagents for amplifying and detecting a second target nucleic acid.