The Principles of ddPCR Technology

Droplet digital PCR has emerged in the scientific community only in the last decade or so. It represents one of the most sensitive, precise, and accurate methods for amplifying and quantifying sequences of different nucleic acids (DNA, RNA, and cDNA).

ddPCR is based on a water-oil emulsion droplet system. The massive partitioning required for this type of PCR is performed in a single step thanks to a combination of surfactants and microfluidic technology. The ideal number of independent partitions or nanoliter-sized droplets in this process is twenty thousand. A single partition should contain zero or one template molecule (but no more than a few).

After the amplification, each droplet is analyzed with a fluorescent probe. If it contains at least one target copy, it is a positive droplet (fluorescent droplet). If it doesn’t contain any target copies, it is a negative droplet (exhibiting little to no fluorescence). This ratio of positive droplets to negative droplets is then analyzed with Poisson statistics to determine the concentration of the DNA template in the original sample.