does the probe have to be within forward and reverse
1 Location: Ideally, the probe should be in close proximity to the forward or reverse primer, but should not overlap with a primer-binding site on the same strand. 2  Probes can be designed to bind to either strand of the target. 3  Melting temperature (Tm): Preferably, probes should have a Tm 6â8Â°C higher than the primers.