KASPar Genotyping of SNPs

The samples were genotyped by LGC Genomics in Hertfordshire. Primer designs for each SNP were prepared using the PrimerPicker software. LGC uses a proprietary genotyping method known as KBiosciences Competitive Allele Specific PCR (KASPar). A separate 5’ sequence was designed as a forward primer for each allele. The reaction mix includes complementary oligonucleotides for these 5’ tails bound either to FAM dye or Victoria fluorescent dye with different excitation and emission wavelengths. The fluorescent dye-bound oligonucleotides are matched by two complementary oligonucleotides bound to quenchers, which reduces the fluorescence emitted. As the DNA is amplified using polymerase chain reaction, more of the fluorescent dye-labelled oligonucleotides are combined, and uncoupled from their quenchers, producing the fluorescence. Each of the two homozygotes then have only one emission wavelength, whereas the heterozygotes has fluorescence at both wavelengths. ROX, also known as carboxyrhodamine, a fluorescent reference dye, was used as a reference to normalise the samples against to account for differences in starting amounts of DNA. Each pair of primers was tested against a panel of 50 samples to check for amplification and dimorphism. Samples were run on 1,384-well plates. Genotype calls were made using KlusterCaller software. Genotyping failed for rs2228570, with no results across the samples tested. This SNP was therefore excluded from the analyses.