PCR takes place within these droplets, leading to the production of a fluorescent readout, either through the use of an intercalating dye or a fluorescent oligomer probe, to indicate the presence or absence of the PCR target of interest. Subsequent quantification of the fluorescent droplets allows a determination of the number of copies of the target locus present in the sample, yielding a typical sensitivity of variant detection in greater than 0.001% of cells (95). Although extremely sensitive, ddPCR requires the optimization of primers, probes, and amplification conditions, which is time-consuming and limits throughput.
