Raw and normalised melt curves. A) Typical examples of raw fluorescence melt curves. Six replicates of an oligonucleotide representing a fragment of the unmethylated CDKN2A promoter were amplified using the CDKN2A primers and QuantiTect SYBR Green PCR Kit, then melted by increasing the temperature from 60 to 90°C, rising in 0.5°C increments, waiting for 30 s at the first step and for 5 s at each step thereafter, acquiring fluorescence at each increment. Raw fluorescence was plotted against temperature. The plots show that the magnitude of the starting fluorescence may vary significantly even between replicates. B) Graph of raw fluorescence plotted against temperature from one sample from Figure 1A shows that there are three distinguishable phases. In Phase 1 there is a linear decrease in fluorescence with increasing temperature (represented as a solid blue line). In Phase 2 there is a rapid reduction in fluorescence which is a combination of the linear decrease and the melting of dsDNA. During Phase 2 the observed fluorescence (F(T)obs) is less than the fluorescence expected from the Phase 1 alone (F(T)max). The reduction in fluorescence due to the melting of the dsDNA alone at any given temperature (vertical dotted red line) is the difference between F(T)max and F(T)obs, F(T)melt (solid red line with arrow heads). In Phase 3 the dsDNA is fully melted and there is no further decline in fluorescence. C) Normalised melt curves for the six replicates in Figure 1A. The normalised fluorescence was calculated by the formula F(T)obs/F(T)max at every given temperature increment. Normalised fluorescence expressed as a percentage was then plotted against temperature.