Fig. 1.

Modified primer design. a The gene and protein locations of the two commons polymorphisms rs6165 and 6166. Although both polymorphisms are present in the same exon, they reside in different protein domains of the receptor. Rs6165 are found in the extracellular domain (ECD) of the receptor, whereas rs6166 resides in the intracellular C-terminal of the receptor. b Mutated and wild-type sequences are amplified simultaneously using a three-primer system, based on the target sequence. c The first primer is designed to amplify only mutated sequences, with one or more melting temperature increasing mutations introduced in the mutated amplicon. The second primer is designed to amplify only wild-type, with one or more melting temperature decreasing mutations introduced in the wild-type amplicon. A third common primer is designed as a reverse primer. d The resulting amplicons differ in nucleotide content according to genotype, which will be reflected in melting temperatures, detectable through melting analysis d. The early peak (67 °C) observed in the negative derivative melting curves for the heterozygous (purple) samples, is due to the formation of heteroduplexes formed between wild-type and mutant amplicons. Figure modified from Casarini et al., 2011 and Kristensen et al., 2012 [15, 26].