Emulsion digital PCR

Project Summary This proposed project aims to develop a new type of digital PCR platform which eliminates the need for uniform volumes and thereby reduces the complexity, cost, and run time of digital PCR while increasing its dynamic range. The result is a higher performance digital PCR system that matches the simplicity, speed, and low per-assay cost of real-time PCR. Real-time PCR maintains great popularity in clinical diagnostics, but digital PCR is superior to real-time PCR because it provides absolute quantitation, greater accuracy at low concentrations, and greater reproducibility. However, because it was hypothesized that uniform volumes are required for digital PCR quantitation, current digital PCR platforms require precise microfluidic chips and control, which result in low throughput and high per-assay costs. Here we propose to address these limitations by developing a digital PCR system that employs variable volume droplets created simply by shaking a sample along with PCR reagents and an oil/emulsifier mix to create an emulsion. PCR is performed, and droplets in the emulsion are imaged and sized. Droplets with one or more copies of a nucleic acid are identified, and nucleic acid concentration is determined. We call this edPCR (emulsion digital PCR). We characterized this edCPR method via computational simulations and validated it experimentally. Simulations were used to investigate the dependence of droplet occupancy on analyte concentration and droplet size distribution, and to estimate the accuracy of the measured concentration in the presence of errors in measurement of droplet volume. Simulations also provided an estimate of dynamic range for a given droplet size distribution and statistical power. The method was validated experimentally in terms of accuracy, precision, and dynamic range, and to rule out potential sources of biased error, such as droplet shrinking and droplet fusion during PCR. In the proposed work, we will develop a commercial edPCR platform that will offer the superior performance of digital PCR while providing the high-throughput (e.g. 384 well plate operation) and low per-assay cost of real-time PCR, which we believe will remove the barriers to the widespread use of digital PCR in clinical assays, point-of-care settings, and large-scale testing.