b) Use of primer or short oligonucleotide specific to the target of interest, as in TaqMan® probes, Molecular BeaconsTM, or Scorpion primers. In case of molecular Beacons, they are labeled with a fluorescent dye or quencher and do not exhibit any significant fluorescence in the free, unhybridized condition. But upon binding to the template, the probe becomes fluorescent as the quencher gets distanced from the fluorescent reporter. The amount of PCR product amplified is directly proportional to the amount of fluorescence.
Fig 3-3.2.2.2: Detection of PCR Product by Molecular Beacon
While in the case of TaqMan® probes, fluorescence occurs when the dye is clipped from the probe during the polymerase extension.
Fig 3-3.2.2.3: explains detection of qPCR product by TaqMan Probe.
[A] In TaqMan probe, when Reporter is adjacent to Quencher –there is no fluorescence.
[B] During extension, polymerase hydrolyzes the probe leading to separation of Reporter from the Quencher and thus fluorescence is exhibited.