Pfcrt Gene Amplification and Sequencing

The DNA from 1 mL blood samples was extracted using a QIAamp™ DNA Blood Midi Kit (QIAGEN), according to the manufacturer’s instructions. PCRs were performed to amplify the pfcrt fragment gene according to previously described protocols [25 (link)]. PCR products were analyzed by electrophoresis on 2% agarose gel and visualized under a UV transilluminator (DigiDoc-It, UVP, Upland, CA, USA). Each PCR product was purified using Wizard™ SV Gel and the PCR Clean-Up System (Promega, WI, USA), following the manufacturer’s procedure. Purified DNA sequencing was carried out through Big Dye™ Terminator Cycle Sequencing Ready Reaction version 3.1 (Applied Biosystems, Carlsbad, CA, USA), with 3.2 μM of forward and reverse PCR primers. DNA sequences to investigate C72S, M74I, N75E/D, and K76T were determined using an ABI Prism DNA Analyzer™ 3730 (Applied Biosystems, CA, USA), on the Fiocruz Genomic Platform PDTIS/Fiocruz RPT01A. Nucleotide sequences were aligned using the ClustalW multiple sequence aligner in the BioEdit software [26 ]. The PF3D7_1343700 strain was used as a reference sequence (from PlasmoDB: http://www.plasmoDB.org, accessed on 20 March 2023). DNA sequences were deposited in GenBank (the NIH’s genetic sequence database; www.ncbi/nlm/nih.gov/GenBank, accessed on 21 March 2023) with the accession numbers OQ672386-OQ672451.