TaqMan

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In molecular biology, quantitative real time PCR methods using a dual-labelled fluorogenic probe called a TaqMan probe is a rapid fluorophore-based real-time PCR method ^[1]. 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 a 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, of dihydrocyclopyrroloindole tripeptide ‘’minor groove binder’’, acronym: MGB) are covalently attached to the 5' and 3' ends of the probe , respectively^[1]. 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 has a lid with built-in fiber optic cables that measure the fluorescence in the reaction tubes (using laser beams for excitation and detection of the fluorescent emission from the fluorophore). Fluorescence intensities are logged and data stored at 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.

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