This investigation demonstrates that digital microfluidic platforms are suitable for automated DNA ligation. Multiple DNA ligation steps are required to create DNA products using oligonucleotide synthesis. Unfortunately, traditional methods of oligonucleotide synthesis are unable to create highly accurate, long DNA products. This leads to a supply-side bottleneck that puts a drag on innovation in drug development, organism engineering, and agricultural improvement. Here we demonstrate ligation of two DNA products into one DNA product in digital microfluidic devices that manipulate droplets in air and in oil. Results from the gel electrophoresis imaging confirmed that ligation on digital microfluidics devices was successful in all cases. Silicone oil experiments also verified that on-chip incubation of DNA ligation is possible on these devices using an external resistive heater. This suggests that large-scale DMF automation of DNA synthesis can be used to alleviate the bottleneck created by the lack of efficient, high-volume production of long change DNA products. Such an advancement would be highly valued for a wide variety of biomedical applications.

Encounters between transcription and DNA replication machineries lead to conflicts that shape genomes, influence evolution, and lead to genetic diseases in humans. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication, increases DNA breaks, and promotes mutagenesis. Here, we show that head-on replication-transcription conflicts lead to pervasive RNA:DNA hybrid formation in Bacillus subtilis. We find that replication beyond head-on conflict regions requires the activity of a RNA:DNA hybrid processing enzyme, RNase HIII. Remarkably, pervasive RNA:DNA hybrid formation at head-on genes completely stops replication and inhibits gene expression in a replication-dependent manner. Accordingly, we find that resolution of head-on conflicts by RNase HIII is crucial for survival upon exposure to various stresses, as many stress response genes are encoded head-on to replication. We conclude that R-loops, RNA:DNA hybrids formed outside of the transcription bubble, exacerbate head-on replication-transcription conflicts, thereby threatening life, especially upon exposure to environmental stresses.