Real time cleavage assay

摘要

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.

主权项

1. A method of sample analysis comprising:
subjecting a reaction mixture comprising reagents for amplifying and detecting a target nucleic acid, comprising:
said target nucleic acid;a set of PCR primers for amplifying said target nucleic acid, wherein said set of PCR primers consists of a single pair of PCR primers that bind to sites in said target nucleic acid;a polymerase activity;a nuclease activity; anda probe that comprises a 5′ fluorophore;to the following thermocycling conditions:
a first set of 5-15 cycles of:
i. a first temperature of at least 90° C.;ii. a second temperature in the range of 60° C. to 75° C.;iii. a third temperature in the range of 65° C. to 75° C.; followed by: a second set of 20-50 cycles of:
i. a fourth temperature of at least 90° C.;ii. a fifth temperature that is at least 10° C. lower than said second temperature; andiii. a sixth temperature in the range of 65° C. to 75° C.; wherein no additional reagents are added to said reaction between said first and second sets of cycles and, in each cycle of said second set of cycles, cleavage of said 5′ fluorophore from said probe by said nuclease activity is measured.