Bead-Droplet Reactor for High-Fidelity Solid-Phase Enzymatic DNA Synthesis

Solid-phase synthesis techniques underpin the synthesis of DNA, oligopeptides, oligosaccharides, and combinatorial libraries for drug discovery. State-of-the-art solid-phase synthesizers can produce oligonucleotides up to 200-300 nucleotides while using excess reagents. Accumulated errors over multiple reaction cycles prevent the synthesis of longer oligonucleotides for the genome scale engineering of synthetic biological systems. The sources of these errors in synthesis columns remains poorly understood. Here we show that bead-bead stacking significantly contributes to reaction errors in columns by analyzing enzymatic coupling of fluorescently labelled nucleotides onto the initiated beads along with porosity, particle tracking and diffusion calculations. To circumvent stacking, we introduce dielectrophoretic bead-droplet reactor (DBDR); a novel approach to synthesize on individual microbeads within microdroplets. Dielectrophoretic force overcomes the droplet-medium interfacial tension to encapsulate and eject individual beads from microdroplets in a droplet microfluidic device. Faster reagent diffusion in droplets, and non-uniform electric field induced enhancement in reagent concentration at its surface can improve reaction fidelities in DBDR. Fluorescence comparisons suggest around 3-fold enhancement of reaction fidelity compared to columns. DBDR can potentially enable the high-purity synthesis of arbitrarily long strands of DNA to meet the emerging demands in healthcare, environment, agriculture, materials, and computing.