2.3.1. Identification of ABCB1 1236C>T (in Exon 12), 2677G>T/A (in Exon 21), and 3435C>T (in Exon 26)

Candidate SNPs (1236C>T, 2677G>T/A, and 3435C>T) in the *ABCB1* gene were genotyped by using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Each primer was prepared according to the variation of allele. Forward primer of 5′ -TATCCTGTGTCTGTGAATTGCC-3′ and reverse primer of 5′ -CCTGACTCACCACACACCAATG-3′ were used for *ABCB1* 1236C>T genotypes. Forward primer of 5′ -TGCAGGCTATAGGTTCCAGG-3′ and reverse primer of 5′ -TTTAG TTTGACTCACCT TCCCG-3′ were used for determination of *ABCB1* 2677G>T/A genotypes. Forward primer of 5′ -TGTTTTCAGCTGCTTGATGG-3′ and reverse primer of 5′ -AAGGCATGTAT GTTGGCCTC-3′ were used for *ABCB1* 3435C>T genotypes. PCR was performed in 20 µL reaction mixture including 1 µL of 10 pmol each primer (forward and reverse), 1 µL extracted DNA, and 17 µL autoclaved distilled water. The PCR program was composed of an initial denaturation at 94 ◦C for 2 min followed by 35 cycles of denaturation at 94 ◦C for 30 s, annealing at 59.8 ◦C for 30 s, and extension at 72 ◦C for 30 s. The final extension step was performed at 72 ◦C for 5 min. The DNA fragments amplified by PCR were reacted at 37 ◦C for 16 h with restriction enzymes *HaeIII* (for *ABCB1* 1236C>T), *Sau3AI* (for *ABCB1* 3435C>T), and *BanI*/*RsaI* (for *ABCB1* 2677G>T/A), which can recognize and cleave specific sequences. These digested fragments were separated by electrophoresis in 2.5% agarose gel, and were visualized under ultraviolet light after staining the gel with ethidium bromide for 30 min.
