Replacement of dhfr-ts gene with a MS/GW construct pDEST/dhfr-ts_1F8Hyg. A) Schematic of the expected genomic loci of dhfr-ts and 1f8Hyg in dhfr-ts+/-/Hyg parasites. B) PCR analysis with gDNA from cloned drug resistant parasites and WT Tulahuen parasites confirm the expected gene deletion of one allele of the dhfr-ts gene and correct insertion of 1f8Hyg. Primer H1 plus the R1, R2 or R3 downstream primers, yield the expected products of 1.8, 2.0 and 2.3 kb, respectively and the combination of H5 plus upstream primers F3, F2 and F1 give the predicted bands of 2.1, 2.4 and 2.8 kb for respectively. See additional file 3: Table S5 for nucleotide sequences of primers. C) Genomic DNA Southern blot analysis of a dhfr-ts+/-/Hyg Tulahuen clone. gDNA digested with BsrGI and hybridized with labeled Hyg CDS probe. Diagram not to scale. Numbers are sizes (bp) of expected products.