Quantifying Malaria Parasite Gene Copy Numbers

pfmdr1 gene copy numbers were determined through real-time PCR using fluorescent TaqMan probe-based gene expression. Primer pairs of pfmdr1 [15 (link)] and the housekeeping gene seryl-t-rna-synthetase were designed to match in length and nucleotide content of the two amplified gene regions. In addition, TaqMan probes with a FAM or VIC dye on the 5′ end and a TAMRA quencher on the 3′ end were added to the reaction mix (Applied Biosystems, USA). The primers used for quantifying copy number were: PF-F: 5′-TTAAGTTTTACTCTAAAAGAAGGGAAAACATA, PF-R: 5′-TCTCCTTCGGTTGGATCATAAAG, PF-FAM: 5′-FAM-CATTTGTGGGAGAATCAGGTTGTGGGAAAT-TAMRA, seryl_F:5′-GATTTATTAAGAAAAATAGGTGGAGCTA, seryl_R: 5′-TATAGCATTATGTAATAAGAAACCTGC, seryl_probe: 5′ VIC-AAGGTATACAAGTAGCAGGTCATCGTGGTT-TAMRA. The reaction mix contained 20 ng DNA, 300 nM primers, 250 nM TaqMan probes, 300 μM dNTPs, 2 mM MgCl₂ and 2.5 units/reaction HotStarTaq DNA Polymerase (Qiagen). Samples were prepared in triplicates. For the reaction, the initial activation step was at 94°C for 3 min., followed by 40 cycles of 94°C for 1 min., 60°C for 1 min. and 72°C for 30 sec. Fluorescence was recorded after each elongation step. Real-time PCR was carried out in a Rotor-Gene RG-3000 (Corbett Research, Toronto, ON, Canada). Copy numbers were determined relative to 3D7, which is known to have only one pfmdr1 gene copy [16 (link),17 (link)].