Extending the Potential of Digital Droplet PCR by Expressing Amplified Genes and Assaying Their Phenotype

Besides its key role in genetic information storage, DNA is also highly relevant to be used as an amplifiable and readable biomarker for plenty of applications including diagnostics, sterility, and quality controls. To this end, several molecular analytical methods emerged over the past decades to specifically detect and quantify DNA sequences from different sample origins. Significant gain in sensitivity was reached with the advent of digital droplet PCR (ddPCR), while also overcoming many important limitations of quantitative PCR. In ddPCR, DNA templates are typically isolated in water-in-oil droplets containing a PCR amplification mixture supplemented with a fluorogenic reporter. Whereas the analytical process usually ends with the counting of the fraction of fluorescent droplets in which PCR amplification took place, in some cases these droplets can be the starting point of more complex analytical pipelines. This is typically the case of the microfluidic-assisted in vitro compartmentalization technology introduced by our team and routinely used to perform in vitro functional screenings and directed evolution experiments. In this chapter, we present this technology in which DNA templates are first amplified by ddPCR, prior to being in vitro expressed as RNA or protein within these droplets. Finally, the phenotype of each gene is analyzed using a fluorescence-based assay and the droplets sorted accordingly.