There are many different kind of primer and probe chemistry systems available for Realtime PCR detection. Only the most prominent ones are exemplarily listed in the figure.
Sybr green DNA intercallating dye
Sybr green binds non specifically to double stranded DNA and fluorescence is increasing with increasing amount of product.
TaqMan Assay
A sequence specific probe carrying a quencher at the 3’ end and a fluorescent reporter at the 5’ end hybridises to the target sequence. During amplification the reporter is cleaved of by the 5’ nuclease activity of the polymerase (therefore also called 5’nuclease assay). As a result the reporter is separated from the quencher and the resulting fluorscence signal is proportional to the amount of amplified product in the sample.
Molecular Beacon
A molecular beacon is a labeled probe that forms a hairpin structure with a stem (5-6 complementary bases) and a loop. A quencher is attached at the 3’ end and a fluorescent reporter at the 5’end. The loop is designed to hybridise specifically to the target sequence. During annealing the molecular beacon binds to its target sequence separating the reporter and the quencher and fluorescence can be
measured. Molecular beacons are only displaced and not destroyed during amplification due to lacking 5’ exonuclease activity of the polymerase. The amount of fluorescence emitted by the reporter is proportional to the amount of target in the reaction.
Hybridisation Probe
Two sequence specific oligonucleotide probes, one carrying a donor fluorophore and one an acceptor fluorophore are designed to bind adjacent sequences in the target. Excitation is performed at the wavelength of the donor fluorophore. The emission spectrum of the donor fluorophore overlaps significantly the excitation spectrum of the acceptor fluorophore. During annealing the hybridised probes are in proximity, allowing fluorescence resonance energy transfer from donor to acceptor. Increasing fluorescence is measured at the emission wavelength of the acceptor fluorophore, which is proportional to the amount of amplicon present.
Scorpion Primer
Scorpion primers are primer and probe in one. They form a hairpin structure with the stem sequence complementary to an internal portion of the target sequence on the same strand. Fluorophore and quencher are at each end with an additional PCR blocker to prevent read through during the extension of the opposite strand. The hairpin structure acts as primer during the first amplification cycle and subsequent denaturation enables the separation of the hairpin leading to hybridisation of the loop sequence to the complementary sequence on the newly synthesised strand. In this position quencher and fluorophore are separated and fluorescence can be measured according to the amplified product.
Amplifluor Primer
Amplifluor primer form a hairpin structure with a quencher at the 5’ and reporter fluorophore at the 3’ end. They can only hybridise to the product from the second amplification cycle having a complementary ‘Z’ sequence origination from the first primer leading to a new target containing the hairpin structure of the primer at the 5’end. During extension of the complementary strand in subsequent cycles this hairpin structure is unfolded leading to fluorescence due to physical separation proportional to the amount of amplified product.
