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 of sample analysis, comprising:
(a) subjecting a PCR reaction comprising a flap endonuclease activity to the following thermocycling conditions:
i. an initial pre-incubation;
ii. 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., wherein all of the cycles are the same; followed by:
iii. 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 comprises a first set of PCR primers for amplifying a target nucleic acid, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites in the target nucleic acid and wherein the target nucleic acid is only amplified by the first set of PCR primers; and
(b) measuring a fluorescent signal in each cycle 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 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 the initial pre-incubation of (a) activates a thermostable polymerase.
6. The method of claim 1 , wherein the method further comprises graphing the amount of fluorescent signal that occurs at 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 each of the second set of at least 20 to 50 cycles, wherein the fluorescent signals respectively indicate the presence of two different mutations.
8. A method of sample analysis comprising:
(a) subjecting a PCR reaction comprising a flap endonuclease activity to the following thermocycling conditions:
i. an initial pre-incubation;
ii. 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., wherein all of the cycles are the same; followed by
iii. 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 comprises a first set of PCR primers for amplifying a target nucleic acid, wherein the first set of PCR primers consists of a single pair of PCR primers that bind to sites in the target nucleic acid and wherein the target nucleic acid is only amplified by the first set of PCR primers; and
(b) measuring a fluorescent signal in each cycle 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 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 the initial pre-incubation of (a) activates a thermostable polymerase.
13. The method of claim 8 , wherein the method further comprises graphing the amount of fluorescent signal that occurs at 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 each of the second set of at least 20 to 50 cycles, wherein the fluorescent signals respectively indicate the presence of two different mutations.