Thermal gradient for fluorometric optimization of droplet PCR in virtual reaction chambers

Abstract

An open system with a thermal gradient is described for the optimization of polymerase chain reactions (PCR). Two thermal electric coolers were used as the heat source. The gradient is measured through encapsulated water-based beads of a temperature-dependent dye inside mineral oil, thereby forming virtual reaction chambers. Nine droplets (with typical volume of 0.7 μL) were used. Using the intrinsic fluorescence of a temperature-sensitive inert dye (sulforhodamine B), the process involves measurement of the fluorescence intensity at a known, uniform temperature together with the instrument-specific calibration constant to calculate an unknown, possibly non-uniform temperature. The results show that a nearly linear thermal gradient is obtained. This gradient function is a useful feature that can be used for optimization of a commonly used enzyme-activated reaction, viz. PCR. The emission spectra of fluorescent droplets during two-step PCR were monitored and the changes in fluorescence between 50 °C and 100 °C quantified. As the gradient feature allows for testing a range of annealing temperatures simultaneously, the optimal annealing temperature can be easily determined in a single experiment.