How do you design a fluorescent hydrolysis probe in qPCR?

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.