Overview of Droplet Digital™ PCR Technology
Droplet Digital PCR (ddPCR™) enables precise, reliable, and highly sensitive quantification of nucleic acids. The technology is based on water-oil emulsion droplet chemistry which fractionates a DNA sample into about 20,000 droplets within a single well. PCR amplification of the template DNA occurs within the individual droplets via specific primers and fluorescent probes, causing the droplets to fluoresce if the target DNA is present. Each droplet is then read in a droplet reader to determine the number of fluorescent and non-fluorescent droplets. These data are analyzed using Poisson statistics to determine the target DNA template concentration in the original sample with extremely high accuracy and precision.
Droplet Digital PCR for Gene Edit Detection
Genome editing has become a widely used tool and allows for specific modification of almost any cell’s genome. However, the often low (<5%) frequency of gene edits, in particular for primary or induced pluripotent stem cells, hinders simple and reliable detection by gel- or sequencing-based methods, or requires laborious clonal isolation of edited cells. Droplet Digital PCR is a powerful technology for ultrasensitive detection and quantification of NHEJ- and HDR-edited alleles in heterogeneous cell pools. This rapid, cost-effective method is amenable to high throughput and provides a convenient readout for the detection of edited alleles and optimization of editing protocols.