Figure 4 | Scientific Reports

Figure 4

From: Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA

Figure 4

Results from optimisation of BRAF V600E (AC > TT) ddPCR assay for annealing temperature, positive template control quantification and assay specificity (cross reactivity) optimisation steps. (a) Using the V600E AC > TT gBlock positive template control in an annealing temperature gradient, optimum separation of negative and positive droplets for both channels was achieved at 59.2 °C (yellow box) and the fluorescence cutoffs indicated (arrows and grey boxes). (b) The V600E AC > TT gBlock 10-fold dilution series (diluted to 7000 copies/ul, 700 copies/ul and 70 copies/ul in Dilution C, D and E, respectively) produced the expected results upon quantification using the previously determined thresholds and (c) there was no significant cross reactivity to any other BRAF V600 mutations in the panel. The arrow highlights low-level cross reactivity to the V600E A > T and V600K gBlocks, below the threshold for positivity. Red circles highlight false positive droplets which were present at acceptably low levels. Cps – copies.

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