For each SNP a region ± 300 bp surrounding the site was downloaded from the UCSC Genome Browser (hg19). SNPmasker 1.0 [31] (hg19 and dbSNP build 132) was used to mask variant alleles in each sequence to avoid introducing allele bias in the PCR primers. PCR primers (Table S10 in Additional file 2) were designed using Primer3. SNaPshot probes (Table S10 in Additional file 2) were designed to anneal adjacent to the mutation site using OligoAnalyzer [32]. Each probe was evaluated for secondary structure formation and designed to have a melting temperature greater than 50°C for the complementary region between the probe and its corresponding template.
