Citation Wadle S, Rubenwolf S, Lehnert M, Faltin B, Weidmann M, Hufert FT, Zengerle R & von Stetten F (2014) Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter. In: Biassoni R & Raso A (eds.) Quantitative Real-Time PCR: Methods and Protocols. Methods in Molecular Biology, 1160. New York: Springer, pp. 55-73. http://link.springer.com/protocol/10.1007/978-1-4939-0733-5_6#; https://doi.org/10.1007/978-1-4939-0733-5_6
Abstract Mediator probe PCR (MP PCR) is a novel detection format for real-time nucleic acid analysis. Label-free mediator probes (MP) and fluorogenic universal reporter (UR) oligonucleotides are combined to accomplish signal generation. Compared to conventional hydrolysis probe PCRs costs can thus be saved by using the same fluorogenic UR for signal generation in different assays. This tutorial provides a practical guideline to MP and UR design. MP design rules are very similar to those of hydrolysis probes. The major difference is in the replacement of the fluorophore and quencher by one UR-specific sequence tag, the mediator. Further protocols for the setup of reactions, to detect either DNA or RNA targets with clinical diagnostic target detection as models, are explained. Ready to use designs for URs are suggested and guidelines for their de novo design are provided as well, including a protocol for UR signal generation characterization.