PrimeTaq™ Probe One-Step RT-qPCR Kit

Detection Principle

Probe-based qPCR uses fluorescent and quencher-labeled DNA probes to target the sequence which will be amplified by PCR. Normally, quenching groups on the probe result in quenching of fluorescent groups due to the fluorescence resonance energy transfer (FRET) in space. When the target sequence is amplified by PCR reaction, both primers and probes are annealed to the target gene. With the extension of primers, the 5' to 3' exonuclease activity of Taq enzyme will cause the probe bound to the target sequence to be degraded gradually from the 5' end. After the fluorescent group and quenching group of the probe are cleaved by Taq enzyme, the quenching group disappears, and the fluorescent group can be normally excited by the excitation light to produce fluorescence. After each PCR cycle, more fluorescent groups are released, and the fluorescence intensity is proportional to the number of newly synthesized target fragments, thus quantitative detection can be achieved. Probes are usually a fragment of linear DNA specific to the target sequence, labeled with fluorescent groups such as FAM or HEX at the 5' end and fluorescent quenching groups such as BHQ1, TAMRA or MGB at the 3' end.