TaqManprobes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications. The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementarytarget sequence and fluorophore-based detection. As in other quantitative PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection. TaqMan probes were named after the videogame Pac-Man as its mechanism is based on the Pac-Man principle.
Principle
TaqMan probes consist of a fluorophorecovalently attached to the 5’-end of the oligonucleotide probe and a quencher at the 3’-end. Several different fluorophores and quenchers are available. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler’s light source via Förster resonance energy transfer. As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals. TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. TaqMan probes can be conjugated to a minor groove binder moiety, dihydrocyclopyrroloindole tripeptide, in order to increase its binding affinity to the target sequence; MGB-conjugated probes have a higher melting temperature due to increased stabilisation of van der Waals forces. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5' to 3' exonuclease activity of the Taq polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the quantitative PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.
Applications
TaqMan probe-based assays are widely used in quantitative PCR in research and medical laboratories:
