Abi prism 3100 dna genetic analyzer

Genotyping of the pfcrt 76 codon (chromosome 7) was performed on both gametocyte and sporozoite DNA with a real-time PCR assay using fluorescence resonance energy transfer (FRET) hybridization probes and a melting curve analysis as previously described (40 (link)). Each run included two control DNA samples of P. falciparum, i.e., the DNA from the CQ-susceptible F32/Tanzania strain corresponding to the pfcrt K76 wild-type allele and that of the CQ-resistant FCM29/Cameroon clone carrying the pfcrt 76T mutant allele.
DNA from gametocytes and DNA from positive salivary glands, after being pooled for each donor, were amplified to assess genetic polymorphism at 7 microsatellite loci located on 5 different chromosomes according to Anderson et al. (41 (link)) and Annan et al. (12 (link)). PCR products were resolved on an ABI Prism 3100 DNA genetic analyzer (Applied Biosystems, Foster City, CA, USA) using 500-LIZ as the internal size standard. Alleles were read under GeneMapper software (Applied Biosystems). The maximum number of alleles at the more polymorphic locus provided the minimum number of clones per isolate and determined the multiplicity of the infection.

Genetic polymorphism was assessed for six microsatellite loci according to Anderson et al. 2000 [9 (link)]. PCR reactions were processed as previously described [2 (link)]. The same protocol was used to amplify DNAs from gametocytes, oocysts and salivary glands, using 2 μl of DNA. PCR products were resolved on an ABI Prism 3100 DNA Genetic Analyzer (Applied Biosystems, Foster City, CA) and alleles were read with the GeneMapper software (Applied Biosystems, Foster City, CA). Multiple alleles were scored automatically and inspected visually to correct allele sizing or artifacts. The MOI was determined as the maximum number of alleles at the more polymorphic locus, which provides the minimum number of clones per isolate as the multilocus genotype of each clone cannot be reconstructed from multiple infections.