To date, nearly twenty hybridization probes have been introduced, all with different fluorescence generation mechanisms. Two commonly applied qPCR probes are highlighted below: the molecular beacon and the TaqMan probe.
Molecular beacons are single-stranded oligonucleotide detector probes that form a stem-and-loop structure. The loop portion of the molecule is a probe sequence that is complementary to a predetermined target sequence and the stem is formed by the annealing of complementary arm sequences that are present on either side of the probe sequence.
A fluorophore is covalently attached to the end of one arm and a nonfluorescent quencher is covalently attached to the end of the other arm. In the absence of target, the stem keeps the fluorophore and the quencher in close proximity to each other, preventing fluorescence (contact quenching). However, when molecular beacons bind to their target they undergo a conformational change that restores the fluorescence of the internally quenched fluorophore.
Molecular beacons have three key properties that enable the design of new and powerful diagnostic assays:
- they only fluoresce when bound to their targets
- they can be labeled with a fluorophore of any desired color
- they are so specific that they can easily discriminate single-nucleotide polymorphisms
As a result molecular beacons enable the development of cost-efficient multiplex diagnostic assays.
Additional information on molecular beacon probes:
Hydrolysis probe (Taqman) assays use a sequence-specific probe labeled with a fluorophore and a quencher. The hydrolysis probe assay uses the 5’-exonuclease activity of the Taq polymerase. When the hydrolysis probe is not hybridized, the 5’-fluorophore will be quenched by the 3’-quencher.
The mechanism of quenching is Fluorescence Resonance Energy Transfer (FRET). However, when the probe is hybridized to the target during the combined anneal/elongation step, the dsDNA-specific 5’-exonuclease activity of the Taq polymerase cuts the hybridized probe. The fluorophore and quencher are no longer on the same molecule and the fluorophore will fluoresce.
Additional information on hybridization probes: