High-throughput Droplet PCR

Abstract
The polymerase chain reaction has facilitated the ready analysis of nucleic acids. The depth of sequence analysis that can be reached via PCR is largely independent of sensitivity requirements, and more a function of the analyte intricacy within the biological sample. A next challenge requires the development of means to unravel the complexity of heterogeneous tissues. Avoidance of bulk measurement has been made possible through adopting principles of digital PCR where samples are diluted to achieve a homogeneous target, which is often presented as a single molecular target. This has presented the task of producing massively parallelized quantitative nucleic acid data from the cellular constituents of tissues. The generation of aqueous droplets in a two phase flow is shown to be readily and routinely facilitated by miniaturized fluidic devices. Droplets serve as ideal means to form discrete packages for the purpose of PCR, offering an enhanced handling potential by virtue of reactant containment, to concurrently eliminate both contamination and sample loss. The packaging into droplets also enables the measurement of nucleic acids from defined populations of cells from bio-samples, or molecules of nucleic acids from the individual nuclei of cells, by means of high throughput, single cell analysis. Details are provided for the production of microfluidic devices for both creating droplets and undertaking PCR in a continuously flowing microfluidic stream. Suggestions are also made as to the optimal fabrication techniques and the importance of device calibration to achieve the conditions favouring efficient PCR.