Applied biosystems 3130 3130xl genetic analyzer

Polymorphisms at position 72–76 in pfcrt and at positions 86, 184, 1034, 1042, and 1246 in P. falciparum multidrug resistance-1 (pfmdr1), which are suggested to be associated with resistance to a variety of anti-malarial drugs [35 (link)], were determined by direct sequencing. An initial and nested PCR were performed with PrimeSTAR Max DNA Polymerase (Takara Bio Inc., Japan) in a 10-μL reaction mixture containing 1 μL of DNA template and 0.5 μM of each primer set. Excess primers and unincorporated nucleotides of the nested PCR product were enzymatically removed using ExoSAP-IT Kit (Amersham Biosciences, Buckinghamshire, UK) and direct sequencing was performed (96 °C or 1 min, 25 cycles of 96 °C for 30 s, 50 °C for 30 s, and 60 °C for 4 min, and 60 °C for 1 min) using a BigDye Terminator v1.1 cycle sequencing kit on the Applied Biosystems 3130/3130xl Genetic Analyzer (Life Technologies, Carlsbad, California, USA). Samples with minor peaks of at least 50% in height compared to the major peak were considered mixed genotypes.
Allele frequencies (proportion of parasite clones in the parasite population that carry a given allele) of drug-resistance genes were estimated using MalHaploFreq [36 (link)], a program that utilizes allele prevalence and MOI data to estimate allele frequencies with a maximum likelihood algorithm using the maximum likelihood methodology.

Plasmodium falciparum DNA was extracted from a quarter of a blood spot (25 µL) using the QIAamp DNA blood Mini Kit (QIAGEN, Hilden, Germany). MOIs or the number of clones per sample were determined by genotyping of merozoite surface protein 2 (msp2), the gene encoding the highly polymorphic locus MSP2, as reported previously [34 (link)]. Briefly, a nested multiplex PCR was performed to amplify 3D7 and/or FC27 family alleles using fluorescence-labelled family-specific primers with Tks Gflex DNA Polymerase (Takara Bio Inc., Japan) in a 10-μL reaction mixture containing 1 μL of DNA template and 0.5 μM of each primer set. The nested PCR products were analysed by 2% agarose gel electrophoresis to select the samples for subsequent capillary electrophoresis analysis. Size variations of nested PCR products were analysed using an Applied Biosystems 3130/3130xl Genetic Analyzer (Life Technologies, Carlsbad, California, USA) and determined with the Peak Scanner software ver2.0 (Thermo Fisher Scientific). If minor peak heights were greater than one-third of the major peak height, these minor peaks were regarded as peaks from minor clones. Samples harbouring two or more alleles were interpreted as multiple-clonal infections.

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.