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How do hydrolysis probes work in qPCR? probes work?

Posted June 22, 2020


Answer

The hydrolysis probe used in qPCR is an oligonucleotide labeled with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end. The background fluorescence of probe is prevented by the presence of the quencher in close proximity. During the annealing step in PCR, both probe and primers anneal to the DNA target. Once Taq polymerase reaches the probe in the extension step, its 5' to 3' exonuclease activity degrades the probe, breaking the reporter-quencher proximity and thus allowing the emission of fluorescence. The increased fluorescence is then used to determine the quantification cycle in each reaction.

Additional resources

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Postollec, F., Falentin, H., Pavan, S., Combrisson, J., & Sohier, D. (2011). Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food microbiology, 28(5), 848-861.

Valasek, M. A., & Repa, J. J. (2005). The power of real-time PCR. Advances in physiology education, 29(3), 151-159.