Digital PCR as a Novel Technology and Its Potential Implications for Molecular Diagnostics

Abstract

The latest incarnation of PCR, digital PCR (dPCR),2 takes 2 decades of development in enzyme chemistry and assay design and applies them with formidable precision and sensitivity. dPCR is achieved by performing a limiting dilution of DNA into a succession of individual PCR reactions (or partitions). Limiting dilution, made practical by advances in partitioning with nanofluidics and emulsion chemistries, capitalizes on the random distribution of the DNA template and the fact that Poisson statistics can be used to measure the quantities of DNA present for a given proportion of positive partitions. And what is more, it appears to work; results obtained with the technique are linear, and it is capable of detecting and quantifying miniscule amounts of template (1, 2). All of these features are achievable without the calibration curve required with almost all other molecular methods for accurately quantifying DNA. Compared with real-time quantitative PCR (qPCR), dPCR has already been heralded as more precise (3), better at detecting rare genetic variants (4), and less susceptible to inhibitors (5, 6). Recognition of these advantages has naturally led to speculation as to the potential of dPCR for molecular diagnostics. This issue of Clinical Chemistry presents the reports of 2 studies that have demonstrated the unique clinical application of dPCR for measuring circulating cell-free nucleic acids. Taly et al. (7) build on their group's leading research in the application of dPCR to investigating the detection of rare tumor-associated mutations in cell-free DNA (cfDNA) in the plasma of cancer patients, and Beck et al. (8) report that the cfDNA of transplantation patients contains detectable quantities of DNA from donor organs and that monitoring of such DNA may serve as a surrogate marker of graft injury and rejection. The articles demonstrate the clinical application of 2 aspects of dPCR, namely …