The new absolute in PCR – Digital PCR

The power of digital PCR – finding a needle in a haystack

Digital PCR (dPCR) is a technique where the sample is partitioned into many individual reactions so that either zero, one or more target molecules are present in each reaction. This approach makes it surprisingly easy to detect a single positive against a strong background of negatives, a typical needle in a haystack problem. It is in such instances that the lower limit of detection excludes standard real-time quantitative PCR (qPCR) as a viable method.

The defining feature of digital PCR is the absolute quantification of nucleic acids. After partitioning, the reactions undergo end-point PCR cycling, and partitions are analyzed for the presence (positive reaction) or absence (negative reaction) of a fluorescent signal. Based on this information, one can calculate the absolute number of molecules present in the sample. Unlike qPCR, dPCR does not rely on standard curves. Thanks to this, dPCR has a lower detection limit and higher precision than qPCR.

Researchers increasingly use digital PCR for sensitive applications, such as copy number variation analysis, gene expression quantification and rare target detection. The method has helped advance cell and gene therapy research, cancer research, infectious disease surveillance, food testing, and many more.