Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR.

Agarose emulsion droplet microfluidic technology for single copy emulsion PCR (ePCR) is a suitable technique for the detection of single copy DNA molecules. It improves the traditional ePCR by employing agarose with low melting and low gelling temperatures, which is coupled with PCR forward primers using Schiff-base reaction. Highly uniform monodisperse nanoliter agarose droplets each containing PCR reagents and single copy template are produced with a microfabricated emulsion generator. Following PCR, the cooled droplets transform to microbeads carrying amplicons to maintain the monocolonity of each droplet, which can be further analyzed. This method allows high-throughput generation of uniform droplets and enables high PCR efficiency, making it a promising platform for many single copy genetic studies.