Probe-based quantitative PCR (qPCR) uses real-time fluorescence from 5ʹ-3ʹ exonuclease cleavage of a fluorescently-labeled, target-specific probe to measure DNA amplification at each cycle of a PCR. Because probe-based qPCR is typically more specific than dye-based qPCR, it is often the foundational technology employed in qPCR diagnostic assays. Probe designs vary but the most common type, hydrolysis (e.g., TaqMan®) probes, incorporate a 5’ reporter fluorophore and a 3’ quencher on a short oligonucleotide complementary to the target sequence. Fluorescence resonance energy transfer (FRET) prohibits emission of the fluorophore while the oligo probe is intact. During each PCR cycle, the 5’ flap endonuclease domain of Taq DNA polymerase hydrolyzes the probe as the primer is extended and the target sequence is amplified. This cleavage event separates the reporter fluorophore from the quencher and results in an amplification-dependent increase in fluorescence. Probe-based qPCR allows multiple targets to be quantified in a single reaction (multiplexing) by using a unique fluorescent dye for each amplicon-specific probe.
TaqMan® is a registered trademark of Roche Molecular Systems, Inc.
SYBR® is a registered trademark of Molecular Probes, Inc.