Probe T m optimization Figure 5 shows how the cleavage rate of probes with different T m s changes with temperature. Probe lengths ranging from a 6mer to a 14mer were investigated and are shown in Figure 5 with calculated T m s of between 14 and 60 C. All the probes showed a bell-shaped relationship for cleavage rate with temperature. As expected, the cleavage rate for all probes increased with increased temperature. A relatively sharp cleavage rate optimum was observed for the probes a, b and c with calculated T m s of 60, 48 and 42, respectively. It appears that the best probe cleavage occurs close to the calculated T m or slightly above it. Performing the assay at a temperature slightly higher than the T m ( Figure 5 ) showed not only optimum activity but also appears to allow probe cleavage cycling, an important feature of this assay.