Representative results for
exon 15 mutation analysis. Sanger sequencing (A - C), high resolution melting (HRM) analysis (D - F), pyrosequencing (Pyro) (G - I) and immunohistochemistry (IHC) (J-L) are compared: The first column shows exemplarily p.V600E mutations, the second p.V600K mutations and the third column p.V600R mutations. In HRM, normalized and temperature shifted difference plots showing wildtype control in blue and mutant control in red. HRM can distinguish between p.V600E (red) and p.V600K (green) and p.V600R (light blue). Pyrosequencing was performed in the reverse direction with the sequence to analyze 5’- YAY TGT AGC TAG ACS AAA AYC ACC -3’. All three mutations can be detected. Immunohistochemistry shows a strong staining for p.V600E (J) but is negative for p.V600K (K) in representative melanoma sample. Pigmentation has to be clearly distinguished from a positive p.V600E staining (K). Cross reactivity was observed for p.V600R mutation (L) in a colorectal carcinoma sample. A: adenine, C: cytosine, G: guanine, T: thymine, V: valine, E: glutamic acid, K: lysine, R: arginine.