Genotyping of Pharmacogenomic Variants

Genomic DNA was extracted from blood samples using the QI Amp DNA mini kit (Qiagen, West Sussex, UK). DNA was quantified using NanoDrop (Thermo Fisher Scientific, Wilmington, DE, USA). Genotyping was conducted using RT-PCR on a DNA Engine Chromo4 system (Bio-Rad Laboratories, Hercules, CA, USA). The PCR procedure consisted of denaturation (95 °C; 10 min), 50 cycles of amplification (92 °C; 15 s) and annealing (60 °C; 1.5 min) [41 (link)]. Taqman genotype master mix and assays, SLC22A6 453G>A (rs4149170, designed using Custom TaqMan® Assay Design Tool) and SLC22A6 728C>T (rs11568626, C__25598602_40) were purchased from Life Technologies (Paisley, Renfrewshire, UK). Opticon Monitor software (v. 3.1, Bio-Rad Laboratories) was used to obtain allelic discrimination plots and identify genotypes. UGT1A1 was genotyped using the Sequenom MassARRAY platform and iPLEX Pro UGT1A1-TA assays (Sequenom Laboratories, San Diego, CA, USA). Similar to methods described by Lee et al. [42 (link)], 20 ng of genomic DNA was amplified by PCR and then treated with shrimp alkaline phosphatase to inactivate unincorporated nucleotides. Using iPLEX Gold Reaction Cocktail, single base extension reaction was performed followed by spotting onto SpectroCHIP II. Data were analysed by MassARRAY TYPER software (v. 4.0.20, Sequenom Laboratories).