Detection of the dhfr 164 SNP alleles: serial dilution of P. falciparum 3D7 genomic DNA. Serial dilutions were performed on the laboratory-adapted strain, 3D7 known to carry the 164 I allele in multiple experiments based on two-fold, five-fold, and ten-fold dilutions. The axes of this plot are in Median Fluorescence Intensity (MFI); x-axis is MFI for 164 I, y-axis is MFI for 164 L. The linear least squares line (solid black; r = 0.82), illustrates how the MFI for 164 L increases as the concentration of 3D7 genomic DNA, and MFI for 164 I increases despite the absence of the 164 L allele in the 3D7 genome. The mean of the background 164 L allele is plotted as a solid black horizontal line; dashed lines designate mean + 1 standard deviation (SD; black), +2 SD (black) and +3 SD (red). Additional experiments focused on the P. falciparum dhfr codon 59 SNP of strains Dd2 and 3D7, and dihydropterate synthetase (dhps) codon 613 SNP routinely showed this same linear increase in background signal associated with increased concentration of the dhfr PCR product.