Digital PCR: a new DNA quantification tool

Digital polymerase chain reaction (PCR) is a quantitative PCR technique with potential to be more accurate and precise than real-time quantitative PCR without the need for a standard curve. The digital PCR sample/reaction mix is randomly distributed into a large number of partitions, such that some partitions contain no copies of the nucleic acid template and others contain at least one copy. Following thermalcycling,partitionscontainingamplifiedproduct can be distinguished by the increased fluorescence generated from the probe. Quantification is achieved by counting the number of positive and negative partitions, followed by Poissonmodelling to account forpartitions containingmore than one template molecule. The partitioning process effectively reduces competition between very similar templates resulting in increased sensitivity for detection of rare mutants in a wild-type background. Digital PCR has been used as a reference method for characterising standards while applications inmicrobiology includemonitoring viral load and detecting bacteria or rare mutant alleles.