RESEARCH ARTICLE Digital PCR Modeling for Maximal Sensitivity, Dynamic Range and Measurement Precision

The great promise of digital PCR is the potential for unparalleled precision enabling accu-rate measurements for genetic quantification. A challenge associated with digital PCR ex-periments, when testing unknown samples, is to perform experiments at dilutions allowing the detection of one or more targets of interest at a desired level of precision. While theory states that optimal precision (Po) is achieved by targeting ~1.59 mean copies per partition (λ), and that dynamic range (R) includes the space spanning one positive (λL) to one nega-tive (λU) result from the total number of partitions (n), these results are tempered for the practitioner seeking to construct digital PCR experiments in the laboratory. A mathematical framework is presented elucidating the relationships between precision, dynamic range, number of partitions, interrogated volume, and sensitivity in digital PCR. The impact that false reaction calls and volumetric variation have on sensitivity and precision is next consid-ered. The resultant effects on sensitivity and precision are established via Monte Carlo sim-ulations reflecting the real-world likelihood of encountering such scenarios in the laboratory. The simulations provide insight to the practitioner on how to adapt experimental loading concentrations to counteract any one of these conditions. The framework is augmented with a method of extending the dynamic range of digital PCR, with and without increasing n, via the use of dilutions. An example experiment demonstrating the capabilities of the frame-work is presented enabling detection across 3.33 logs of starting copy concentration