Real Time Cleavage Assay
AssigneeExact Sciences Corporation
InventorsRebecca Oldham-Haltom, Hongzhi Zou, Graham P. Lidgard, Michael J. Domanico, Hatim Allawi
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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.
Claims16
1-13. (canceled)
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.
Dependencies are inferred from the claim text.