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How do you design a fluorescent hydrolysis probe in qPCR?
Posted June 22, 2020
Answer
The fluorescent hydrolysis probe endows qPCR with high specificity, which usually have the following properties:
- Length: The probe should be shorter than 30 nucleotides, otherwise an internal quencher is needed to ensure low background.
- Location: The probe is ideally in close proximity to the primer, either the forward primer or the reverse primer, but should not overlap with a primer-binding site. Probes can be designed to bind to either strand of the target.
- Melting temperature (Tm): Probes should have a melting temperature 6-8 °C higher than the primers. A too low Tm can result in compromised sensitivity because of the low percentage of probe bound to target.
- GC content: Probes should have a GC content of 35-65% and avoid a G at the 5’ end to preventing quenching of the 5’ fluorophore.
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.