TaqMan probes (also known as Fluorogenic 5’ nuclease assay) contain two dyes, a reporter dye (e.g. 6-FAM) at the 5’ end and a 3’ acceptor dye, usually TAMRA. Recent designs substitute the 3’ TAMRA fluorescent acceptor quencher dye with non-fluorescent quencher, e.g. Black Hole Quencher. The proximity of the quencher to the reporter in an intact Taqman probe allows the quencher to suppress, or “quench” the fluorescence signal of the reporter dye through FRET. If the target of interest is present, these Taqman probes specifically anneal between the forward and reverse primer sites. During the reaction, the 5’ to 3’ nucleolytic activity of Taq polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are displaced from the target, separating the reporter dye from the quencher dye and thus resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. Because increase in fluorescence signal is detected only if the target sequence is complementary to the probe, nonspecific amplification is not detected.
Design of TaqMan Probes and Primers
Some guidelines for TaqMan probes and primers selection are as follows:
G-C content between 20% and 80%
Avoid runs of an identical nucleotide, especially guanine
Avoid G to be on the 5’ end
Probes and primers should contain more C than G
Melting temperature (Tm) should be 68-70°C for probes and 58-60°C for primers
The five nucleotides at the 3’ end of each primer should have no more than two Gs and/or Cs
Give precedence to better probes over primers
Probe should be as close to 5’ primer as possible without overlapping
