Real time cleavage assay
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. A method for detecting a target nucleic acid, comprising:
(a) subjecting a PCR reaction mixture comprising a flap endonuclease activity to thermocycling conditions that comprise:
i. a first set of 5 to 15 cycles each comprising a denaturation step followed by one or more steps in which all temperatures are in the range of 60° C. to 75° C.; followed by:
ii. a second set of 20 to 50 cycles each comprising a denaturation step followed by one or more steps comprising a temperature that is in the range of 50° C. to 57° C.;
wherein the PCR reaction mixture comprises at least a first set of PCR primers, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites that flank the target nucleic acid and wherein the target nucleic acid is amplified by the first set of PCR primers and not any other primers that may be present in the PCR reaction mixture; and
(b) detecting a fluorescent signal in at least some cycles of the second set, wherein the fluorescent signal is generated by cleavage of a probe by the flap endonuclease activity and indicates the presence of a target nucleic acid
wherein no additional reagents are added to the PCR reaction mixture between said first and second sets.
2. The method of claim 1 , wherein the cycles of the second set of 20 to 50 cycles each comprises a temperature that is in the range of 50° C. to 55° C.
3. The method of claim 1 , wherein the measuring step of (b) comprises measuring cleavage of a fluorophore from an oligonucleotide in each of the second set of 20 to 50 cycles.
4. The method of claim 1 , wherein the fluorescent signal of (b) indicates a mutation.
5. The method of claim 1 , wherein step (a) comprises an initial pre-incubation step that activates a thermostable polymerase.
6. The method of claim 1 , wherein the method further comprises graphing the amount of fluorescent signal that is detected in each of the 20-50 cycles, thereby providing an estimate of the abundance of the target nucleic acid in the reaction mix.
7. The method of claim 1 , wherein step (b) comprises measuring two fluorescent signals in at least some of cycles of the second set, wherein the fluorescent signals respectively indicate the presence of two different mutations.
8. A method for detecting a target nucleic acid comprising:
(a) subjecting a PCR reaction mixture comprising a flap endonuclease activity to thermocycling conditions that comprise:
i. a first set of 5 to 15 cycles each comprising a denaturation step followed by one or more steps in which all temperatures are in the range of 65° C. to 75° C.; followed by
ii. a second set of 20 to 50 cycles each comprising a denaturation step followed by one or more steps comprising a temperature that is in the range of 50° C. to 65° C.,
wherein the PCR reaction mixture comprises at least a first set of PCR primers, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites that flank the target nucleic acid and wherein the target nucleic acid is amplified by the first set of PCR primers and not any other primers that may be present in the PCR reaction mixture; and
(b) detecting a fluorescent signal in at least some cycles of the second set, wherein the fluorescent signal is generated by cleavage of a probe by the flap endonuclease activity and indicates the presence of a target nucleic acid, wherein no additional reagents are added to the PCR reaction mixture between said first and second sets.
9. The method of claim 8 , wherein the cycles of the second set of 20 to 50 cycles each comprises a temperature that is in the range of 50° C. to 55° C.
10. The method of claim 8 , wherein the measuring step of (b) comprises measuring cleavage of a fluorophore from an oligonucleotide in each of the second set of 20 to 50 cycles.
11. The method of claim 8 , wherein the fluorescent signal of (b) indicates a mutation.
12. The method of claim 8 , wherein step (a) comprises an initial pre-incubation step that activates a thermostable polymerase.
13. The method of claim 8 , wherein the method further comprises graphing the amount of fluorescent signal that is detected in each of the 20-50 cycles, thereby providing an estimate of the abundance of the target nucleic acid in the reaction mix.
14. The method of claim 8 , wherein step (b) comprises measuring two fluorescent signals in at least some of the cycles of the second set, wherein the fluorescent signals respectively indicate the presence of two different mutations.