Abi 3100 genetic analyser

Cultures of “M. nasistruthionis sp. nov.” str. Ms03 (GenBank: KM410300.1) were obtained from Mr J.J. Gouws (Faculty of Veterinary Science, Onderstepoort, University of Pretoria). Genomic DNA (gDNA) was isolated from these cultures using a method described by Hempstead (29 (link)). The Type A oppA gene (24 (link)) was amplified from gDNA and cloned into the pGEM®-T Easy vector (Promega). Primers used are listed in Supplementary Table 1 and PCR conditions described in the Supplementary Procedures. To allow eukaryotic expression of the oppA gene, it was subjected to site-directed mutagenesis (SDM) to change 16 mycoplasma tryptophan codons (TGA) to universal tryptophan codons (TGG). The PCR primers used for SDM are listed in Supplementary Table 1 and the SDM procedure outlined in Supplementary Procedures. Correct mutation of the gene was confirmed with sequencing by the Central Analytical Facility, DNA Sequencing Unit of Stellenbosch University using an ABI® 3100 Genetic Analyser (Applied Biosystems, USA).

Nine short tandem repeat (STR) markers (ARAII, PfPK2, poly-alpha, TA1, TA42, TA60, TA81, TA87 and TA109) described by Anderson et al were used to genotype P. falciparum isolates [34 (link)]. For P. vivax, a panel comprising eight STR markers (MS1, MS5, MS10, MS12, MS20, MS16, msp1F3, and PV3.27) described by Koepfli et al and Karunaweera et al. were used [35 (link), 36 (link)]. The primers and PCR conditions for the assays are described elsewhere [37 (link)]. The labelled PCR products were sized on an ABI 3100 Genetic Analyser with GeneScan LIZ-600 size standard (Applied Biosystems). The resulting electrophoretograms were analysed using the online, open-access vivaxGEN platform [38 (link)]. All genotypes can be accessed in vivaxGEN. The P. vivax genotypes are available under the batch codes IDPV-XXV, IDPV-TES, IDPV-ACT and IDPV-ACT2, and the P. falciparum genotypes are available under IDPF-XXV, IDPF-TES, IDPF-ACT and IDPF- ACT2. An arbitrary intensity threshold of 100 relative fluorescence units (RFU) and minimal 33% peak intensity of minor relative to predominant peaks was used to reduce background noise/artefacts. Only samples with information in at least 50% of the loci were considered successfully genotyped.

Plasmodium falciparum genotyping was conducted using nine previously described short tandem repeat (STR) markers (ARAII, PfPK2, poly-alpha, TA1, TA42, TA60, TA81, TA87, and TA109), following the protocol described by Anderson et al.^{20 (link)} For P. vivax genotyping, nine previously described STR markers selected as a consensus panel by the Asia Pacific Malaria Elimination Network Vivax Working Group (Pv3.27, MS16, MS1, MS5, MS8, MS10, MS12, MS20, and msp1F3) were used following previously described protocols.^{21 (link),22 (link)} The fluorescently labeled PCR products were separated by capillary electrophoresis on an ABI 3100 Genetic Analyser with GeneScan LIZ-600 size standard (Applied Biosystems, Mulgrave, Victoria, Australia). The resulting electrophoretograms were analyzed using VivaxGEN version 1.0 and verified manually.^{23 (link)} To reduce potential artifacts from background noise, an arbitrary fluorescent intensity threshold of 50 relative fluorescence units was used. Only samples with information in at least 50% of the loci were considered as successfully genotyped.

DNA obtained from matched normal and tumor samples was amplified using specific primers. The purified products were sequenced using BigDye terminator v3.1 (Applied Biosystems, Foster City, CA) with ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA).

RT-PCR was performed using 2 μl cDNA in a standard 50 μl reaction set up containing: 2X “KOD hot start" master mix, and 10 μM each forward and reverse primers. The forward primer was fluorescently-labeled with 6—FAM (carboxyfluorescein) at the 5' end. PCR conditions were 2 min at 95°C, followed by 35 cycles of 20 sec at 95°C, 10 sec at the annealing temperature, and 15 sec at 70°C. The RT-PCR products were mixed with Hi-Di Formamide (Applied Biosystems) and an internal size standard (GeneScan -500 Rox, Applied Biosystems). Products were separated by capillary electrophoreses on an ABI 3100 Genetic Analyser using POP4 polymer (Applied Biosystems) and analyzed with the Gene Mapper Software Version 3.7 (Applied Biosystems). Primer sequences were CFTR-e11-Forward: /56-FAM/ATT TCA TTC TGT TCT CAG TT, CFTR-e13-Reverse: 5’- TCA GCA TCT TTG TAT ACT GC-3’.
Automated sizing of DNA fragment was performed by the electrophoresis of RT-PCR product on Fragment Analyzer Automated CE System using 35 bp-1500 bp size standards available from Advanced Analytical Technologies.