Droplet Digital PCR for Mutation Quantification

The final concentration of digested DNA was adjusted to yield less than ∼3500 positive molecules per μl, which is within the range of linearity for the Poisson calculation (38 (link)). Reaction mixtures (25 μl) contained ddPCR Master Mix (Bio-Rad, Hercules, CA, USA), 250 nM TaqMan probe, 900 nM of each appropriate flanking primer and 0–100 ng of TaqI-digested DNA. Reaction droplets were made by applying 20 μl of each reaction mixture to a droplet generator DG8 cartridge (Bio-Rad) for use in the QX100 Droplet Generator (Bio-Rad). Following droplet generation, 38 μl of the droplet emulsion was carefully transferred to a Twin.tec semi-skirted 96-well PCR plate (Eppendorf, Hamburg, Germany), which was then heat-sealed with a pierceable foil sheet. To amplify the fragments, thermal cycling was carried out using the following protocol: initial denaturation step at 95°C for 10 min, followed by 40 cycles of 94°C for 30 s and 58°C for 1 min. The thermally cycled droplets were analyzed by flow cytometry in a QX100^TM Droplet Digital^TM Reader (Bio-Rad) for fluorescence analysis and quantification of mutation frequencies.