Figure 8-11 Fluorogenic probes (probe with an attached fluorophore, a fluorescent molecule that can absorb light energy and then is elevated to an excited state that is released as fluorescence in the absence of a quencher) commonly used for detection of amplified product in real-time PCR assays. A, Hydrolysis probe. In addition to the specific primers for amplification, an oligonucleotide probe with a reporter fluorescent dye (R) and a quencher dye (Q) at its 5' and 3' ends, respectively, is added to the reaction mix. During the extension phase, the quencher (molecule that can accept energy from a fluorophore and then dissipate the energy so that no fluorescence results) can only quench the reporter fluorescence when the two dyes are close to each other, which is only the case with an intact probe (a). Once amplification occurs and the fluorogenic probe binds to amplified product, bound probe is degraded by the 5'-3' exonuclease activity of Taq polymerase; thus quenching is no longer possible and fluorescence is emitted and then measured
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