Genotyping of Antimalarial Resistance Genes

Polymorphisms at amino acid positions 72–76 in the P. falciparum chloroquine resistance transporter gene (pfcrt) were determined by direct sequencing. In the P. falciparum multidrug resistance-1 (pfmdr1) gene, polymorphisms at codons 86, 184, 1034, 1042 and 1246 were determined by direct sequencing and/or restriction fragment length polymorphism (RFLP) analysis, as previously described [29 (link), 32 (link)]. For direct sequencing, initial and nested PCR was done with PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Japan) in 10 μL reaction mixture containing 1 μL of DNA template and 0.5 μM of each primer set. Excess primers and unincorporated nucleotides from the nested PCR product were enzymatically removed with ExoSAP-IT Kit (Amersham Biosciences, Buckinghamshire, UK) and direct sequence was performed (96 °C for 1 min, 25 cycles of 96 °C for 30 s, 50 °C for 30 s and 60 °C for 4 min, and a final cycle at 60 °C for 1 min) with a BigDye Terminator v1.1 cycle sequencing kit in the Applied Biosystems 3130/3130xL genetic analyzer (Life Technologies, Carlsbad, California, USA). Samples with overlapping peaks of at least 50% in height were considered harboring mixed genotypes.