Fig. 1
A typical HRM plot is depicted. The LightCycler® 480 Gene scanning software v1.5 automatically characterizes samples according to the HR melting pattern of the amplified DNA. a shows the normalized curve of fluorescence change in each small division of temperature change imparted onto the assay mix. Fluorescence of a double stranded DNA is taken as 100% and that of a fully denatured DNA is assigned as 0%. The temperature range at which the melting curve is analyzed is adjusted to align all the samples, according to the Tm, to obtain uniform values of post- and pre-melt stages. Once the normalization is done, the software automatically differentiates samples that have a shifted melting curve from wild type. In the example above, the blue curves depict samples with wild-type DNA (wtDNA) while the red represents those with mutant DNA (mutDNA). The wtDNA has a slope (i.e. dF/dT) different from that of mutDNA. The second plot (b) is the “Difference plot”. In this graph, dF/dT (y-axis) is plotted against melting temperature range (x-axis), by subtracting the normalized shifted curve from a normalized base curve. The difference plot can visually differentiate even small changes in fluorescence intensity in unit temperature change. For this assay, 5 ng of total DNA was included to replicate previously established HRM assay condition