TaqMan

] . The TaqMan Real-time PCR measures accumulation of a product via the fluorophore during the exponential stages of the PCR, rather than at the end point as in conventional PCR. The exponential increase of the product is used to determine the threshold cycle, C_T, i.e. the number of PCR cycles at which a significant exponential increase in fluorescence is detected, and which is directly correlated with the number of copies of DNA template present in the reaction.

The set up of the reaction is very similar to conventional PCR, but is carried out in a real-time thermal cycler that allows measurement of fluorescent molecules in the PCR tubes. Different from regular PCR, in TaqMan real-time PCR a "probe" is added to the reaction, i.e., a single-stranded oligonucleotide complementary to a segment of 20-60 nucleotides within the DNA template and located between the two primers. A fluorescent reporter or fluorophore (e.g. 6-carboxyfluorescein, acronym: "FAM", or tetrachlorofluorescin, acronym: TET) and "quencher" (e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide "minor groove binder", acronym: MGB) are covalently attached to the 5'- and 3'-ends of the probe, respectively [cite journal |author=Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J|year= 2000|title=3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures|journal=Nucleic Acids Res|volume=28|pages=655–661|pmid=10606668|doi=10.1093/nar/28.2.655] .

The close proximity between fluorophore and quencher attached to the probe inhibits fluorescence from the fluorophore. During PCR, as DNA synthesis commences, the 5' to 3' exonuclease activity of the Taq polymerase degrades that proportion of the probe that has annealed to the template (hence its name: Taq polymerase + PacMan). Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

Real Time Thermal Cycler

A real-time PCR cycler measures the fluorescence in the reaction tubes (using laser beams or LEDs for excitation of the fluorophore). Fluorescence intensities are logged and data stored for each PCR cycle and then used to create amplification plots of ΔRn (fluorescent signal detected - background) vs cycle number to identify the threshold cycle, C_T, which is used to quantitatively determine the amount of DNA template present in the PCR.

References