CHAPTER 5: Digital Polymerase Chain Reaction (dPCR) – General Aspects and Applications

The application of digital polymerase chain reaction (dPCR) for the detection and absolute quantification of DNA is steadily increasing in various fields, including medicine, bacteriology, virology, agriculture and food analysis. In contrast to quantitative real-time PCR (qPCR), DNA can be absolutely quantified with high precision without the need for external calibrants (standard curves). In dPCR, the PCR mix is partitioned into a large number of small partitions (up to millions) of a very small volume (down to a few picoliters). These can be either physically distinct compartments on a chip [chamber-based dPCR (cdPCR)], or these compartments correspond to water-in-oil droplets [droplet digital PCR (ddPCR)]. Common to both approaches, the number of partitions classified as either positive (target DNA detected) or negative (target DNA not detected) is counted at the end-point of the reaction by fluorescence measurement. The term “digital” refers to these individual positive (“1”) or negative (“0”) PCR results. By statistical analysis of the results the absolute quantity of target DNA in a sample can be determined.