Figure 2

SND-ddPCR assay development and optimization. (a) Schematic representing primer/probe annealing and optimization strategy for the SND-ddPCR assay. Left: F and R indicate shared forward and reverse primers, respectively. Probes 1 and 2 are hydrolysis probes with FAM (blue) or HEX (green) fluorophores on the 5′ (left) end, and a quencher on the 3′ (right) end. Middle: Sequence of probes used to detect albino or WT allele. Internal ZEN quencher (Z) was used to reduce background. A bold letter indicates single-base difference between probes. Right: Theoretical melt curve for hydrolysis probes. The solid line represents probe annealing when all bases are matched to the target; the dashed line represents annealing with a single base mismatch. The ideal temperature range for SND assays is highlighted in yellow, which results in maximum separation between matched and mismatched annealing. (b–d) 2D display of SND-ddPCR results. Clusters composed of positive or negative microreactions are shown as a heat map in each quadrant. CD1 (1362 copies/µl), BL6 (1353 copies/µl) and CD1 + BL6 mixture (combined 2719 copies/µl) are shown in (b), (c), and (d), respectively. (e) 2D display showing centroid of the Tyr^Alb, Tyr^WT, and the double-positive cluster when analyzed at various annealing temperatures. The highest temperature is shown at the bottom left, and the lower temperatures move along the respective line upward and/or rightward. (f) Frequency of Tyr^Alb and Tyr^WT allele detected in various mouse strains. CD1xDBA2J is an F1 hybrid.