Genotyping of CYP2D6*10.
Peripheral blood samples were collected and DNA was extracted by total genomic
DNA isolation using the Tianamp Blood DNA Kit (Tiangen Biotech Beijing Co., Ltd.,
China). Three alleles – CYP2D6*1/*1, CYP2D6*1/*10, and CYP2D6*10/*10 – were
identified using polymerase chain reaction (PCR).
The sequence of the sense primer was 5’-GAGGAGCCCATTTGGTAGTGA-3’
and the sequence of the antisense primer was 5’-TGGTCCAGCCTGTGGTTTC-3’.
The 20-μL PCR reaction mixture contained 7 μL PCR RNase-free water, 10 μL 2×
TAQ master Mix (TOYOBO Co. Ltd., Japan), 1 μL of each primer, and 1 μL of
genomic DNA sample. The PCR process was performed using a PCR amplifier
(ABI/SCIEX Co., Ltd., USA). The PCR reaction conditions were as follows: initial
denaturation for 5 min at 94ºC, followed by 35 cycles of 30 s at 94ºC, 10 s at 55ºC,
and 30 s at 72ºC, and then a final terminal extension of 7 min at 72ºC. The products (2
μL) were analyzed by 1.5% agarose gel electrophoresis and sequenced using an ABI
3730 Genetic Analyzer (Applied Biosystems, USA). The CYP2D6*10 genotypes
were confirmed using the Chromas program (Technelysium, Australia).