Opticon monitor v 3

Genomic DNA was extracted from whole blood through use of the manufacturers protocol (E.Z.N.A Blood DNA Mini Kit; Omega bio-tek; Norcross, GA). Extracted DNA was quantified using NanoDrop (Thermo Fisher Scientific, Wilmington, DE). Genotyping was completed using real-time allelic discrimination polymerase chain reaction (PCR) assay on a DNA Engine Chromo4 system (Bio-Rad Laboratories, Hercules, CA). The PCR protocol followed denaturation at 95°C for 10 minutes, followed by 50 cycles of amplification at 92°C for 15 seconds and annealing at 60°C for 1 minute 30 seconds. Taqman Genotyping Master mix and assays CYP2B6 516G>T (rs3745274, catalogue number C___7817765_60), CYP2B6 983T>C (rs28399499, catalogue number C__60732328_20), CYP2B6 15582C>T (rs4803419, catalogue number C___7817764_10), CYP2A6 -48A>C (rs28399433, catalogue number C__30634332_10) CYP2A6*9B 1836G>T (rs8192726, catalogue number C__29560333_20) NR1I2 63396C>T (rs2472677, catalogue number C_2607845), NR1I3 540C>T (rs2307424, catalogue number C__25746794_20) and NR1I3 1089T>C (rs3003596, catalogue number C__16194070_10) were purchased from Life Technologies (Paisley, Renfrewshire, UK). Opticon Monitor v.3.1 software (Bio-Rad Laboratories) was used to obtain allelic discrimination plots and identify genotypes.