2.2. Sequence acquisition and analysis

Total genomic DNA was extracted using an adapted phenol-chloroform protocol [37]. We then sequenced a 537 base pair (bp) region of the MC1R coding region using the primers 5'-ctggagatgggtgcttcttc-3′ and 5'-tctttgtagccatgctggtg-3′ as described in detail by Peters et al. [34]. Briefly, purified PCR products were sequenced in both directions using the Applied Biosystems BigDye Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific: Waltham, MA, USA) and analysed on an ABI 3730xl capillary sequencer. The laboratory work was performed at Bielefeld University. Consensus sequences were then generated using ChromasPro v. 1.3.4 and aligned manually within BioEdit v. 5.0.6. The resulting alignment was used to quantify the frequencies of the wild-type and mutant (S291F) allele in each population. Heterozygous sites were identified as those with two peaks of roughly equal intensity but around half the intensity of a homozygote.