Figure 4.

Melting temperature (T_m), maximum T_m, ΔT_m. The T_m was defined as the temperature at which the probe's fluorescent signal intensity was 50% of that of the probe with the highest signal intensity in the same probe set. Probes that were <50% hybridized at 45°C were assigned a T_m of 45°C. For each probe set, the maximum T_m was defined as the highest T_m of the six probes (one for each K103 codon) with the same flanking sequence. The ΔT_m was defined as the absolute value of the difference between the maximum T_m for the probe set to which the probe belonged and the T_m of the probe. By definition, the probe with the maximum T_m has a ΔT_m of 0°C, whereas the remaining five probes in a set would have a ΔT_m > 0°C. In each experiment, the mean and median ΔT_m across each of the three probe sets were calculated for each of the six codon 103 variants.