Dimethyl sulfoxide (dmso)

Specific primers were provided by PHUSA Biochem Company (Can Tho, Vietnam; Appendix 1). Polymerase chain reaction (PCR) was performed using a total reaction volume of 20 µl that included 10 ng genomic DNA, 1X NEB master mix (New England Biolabs, Ipswich, MA), 10 pmol of each primer, 1 µl DMSO (Bioworld, Dublin, Ohio (OH; 43017), and 16.5 µl deionized water. The thermal cycle comprised denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, 68°C for 20 s, and a final extension at 68°C for 5 min. The PCR products were then purified using a MultiScreen-PCR 96 Filter Plate (Merck-Millipore, Burlington, MA) and subsequently bi-direction sequenced using an ABI Prism BigDye Terminator Cycle Sequencing Kit Version 3.1 (Applied BioSystem, Waltham, MA) on an ABI genetic analyzer 3500 (Applied Biosystems, Waltham, MA).

Sanger sequencing method was used to identify nucleotide variants in the promoter, entire 9 coding exons and their flanking regions of CYP2D6 gene. Primers provided by PHUSA Biochem Company (Can Tho, Vietnam) were listed in Supplementary Table 1. Polymerase chain reaction (PCR) reaction was performed with a total volume of 20 μL containing: 10 ng total genomic DNA, 1× DreamTaq Master mix (Thermo Fisher Scientific, Waltham, Massachusetts), 10 pmole each primer, 1 μL DMSO (Bioworld, Dublin, Ohio), and 16.5 μL deionized water. The thermo cycle was as following: denaturation at 95 °C for 5 minutes, following by 40 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, 72 °C for 40 seconds to 2 minutes, and a final extension at 72 °C for 5 minutes. For exon 5 to exon 8, the thermo cycle was as following: denaturation at 95 °C for 2 minutes, following by 35 cycles of 95 °C for 30 seconds, 59 °C for 30 seconds, 72 °C for 2 minutes, and a final extension at 72 °C for 5 minutes. PCR products were purified using Multiscreen PCR 96 Filter Plate (Merck-Millipore, Burlington, Massachusetts) and were then sequenced using ABI Prism BigDye Terminator Cycle Sequencing Kit Version 3.1 (Applied Biosystems), on an ABI genetic analyzer 3500 (Applied Biosystems).