Figure 1

DMAS qPCR principle. Each DMAS-qPCR assay consists of a common primer (grey) and allele-specific primers targeting either the REF (green) or ALT (red) allele. Allele-specific primers are located with their 3′ terminal end on the SNP of interest and include an additional mismatch at the fourth nucleotide from the 3′ terminal end. For each allele-specific primer, an individual PCR reaction is performed, in combination with the common primer in parallel. Depending on the genotype status of the sample, both (heterozygous sample) or only one of the allele-specific primers (homozygous sample) will result in elongation and the generation of a signal. (B) By combining the signal status of both reactions, the genotype status of the sample can be deduced. (C) In models 1 and 2, this is achieved using the difference in Cq values from both reactions/signals (heterozygous samples will result in a dCq value approximating zero). In model 3, the Cq values of both reactions are plotted on opposite sides of a scatter plot, followed by genotype calling by means of clustering (D).