T. cruzi TcSC5D gene encodes a putative C-5 sterol desaturase (TcCLB.473111.10, TcCLB.507853.10), this was amplified by PCR followed by direct sequencing to determine DTU-specific genotypes using eight key discriminating single nucleotide polymorphisms (SNP’s) as previously described [64 (link)]. Briefly, a 832bp TcSC5D fragment was amplified under the following PCR conditions: 50 pmol TcSC5D-forward (5´-GGACGTGGCGTTTGATTTAT-3´) & reverse primers (5´-TCCCATCTTCTTCGTTGACT-3´), 100 ng genomic DNA, Platinum PCR Supermix High Fidelity (ThermoFisher Scientific) in a final volume of 50 μl. Samples were denatured at 94°C for 5 min, followed by 35 cycles at 94°C 30 s, 58°C 30 s, 72°C 30 s, and extension at 72°C for 5 min. Amplification products were visualised on 1.2% agarose with ethidium bromide as a single 832 bp product, and subsequently sequenced with the same PCR primers using Applied Biosystems 3730 DNA Analyser. Chromatograms were analysed using Chromas software and alignments generated using Clustal Omega to identify nucleotides at key position 138, 168, 336, 495, 618, 648, 657 and 747 and determine DTU assignment TcI-VI (S1 Table).
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