Genotyping assays

DNA extraction from whole blood was completed by the non-enzymatic, high-salt procedure as described by Lahiri and Nurnberger [41 (link)]. We tested the functional A118G single nucleotide polymorphism (SNP), which causes a missense amino acid change from an aspartate residue to an asparagines residue, thus potentially removing an N-glycosylation site [42 (link)]. This SNP was genotyped using commercially available genotyping assays (Applied Biosystems Inc., Foster City, CA, USA). Genomic DNA (20 ng) was amplified in 10-μL reactions by polymerase chain reaction with the following conditions: 95 °C 10 min, followed by 50 cycles of 92 °C 15 s, 60 °C 1min. The Allelic Discrimination Program on ABI7000 Prism Sequence Detection System was used to determine the genotypes of each individual. Genotypes were tested for fitness to Hardy-Weinberg Equilibrium using Haploview version 4.2 (Broad Institute, Cambridge, MA, USA) [43 (link)].

Genomic DNA was isolated from white blood cells using the Gentra Puregene Blood Kit according to the manufacturer's instructions. Reagents for TaqMan drug metabolism Genotyping Assays for allelic discrimination (Applied Biosystems Genotyping Assays) were used to establish the genetic profile of study participants. The following assays identification numbers were used: C_7817765_60 rs3745274 for CYP2B6, C_1837671_50 rs2740574 for CYP3A4 rs2740574, a custom designed TaqMan assay was performed for CYP2B6 rs2279343. The 7500 Fast Real-Time PCR System (Applied Biosystems) was used to genotype the different SNPs. PCR mixture consisted of a 5 μl TaqMan master mix (2X), 0.5 μl TaqMan drug metabolism Genotyping Assays mix (20X), and 1 μl of DNA completed to 10 μl with nuclease free water. The PCR run method was as follows: an initial step at 60°C for 30 s, hold stage at 95°C for 10 min followed by 40 cycles: step 1 at 95°C for 15 s and step 2 at 60°C for 1 min supplemented by a read stage at 60°C for 30 s.