Fluorescence Microscopy Visualization of DNA Breakage in Extensional Flows of Microfluidic Devices

Abstract

Scission of DNA into smaller fragments is an important step in next-generation sequencing, with hydrodynamic breakage being a preferred method. Previous studies on flow-induced scission of DNA (and polymers in general) have focused on observing the distribution of scission products to infer the breakage mechanism and kinetics. Few studies have directly visualized the breakage of individual molecules in a flow of controlled type.We employed fluorescence microscopy to image the breakage dynamics of labelled DNA molecules in the extensional flow of microfluidic devices. By observing the distribution of breakage locations for 29 molecules and comparing breakage probability in extensional versus channel flows, we reveal that mechanical stretching at the tested conditions does not sufficiently weaken the backbone bond, and breakage is caused solely by photocleavage. Ligated DNA molecules broke less frequently than those from the stock solution, indicating that nicking increases the breakage probability. Increasing the mechanical stretching force through viscosity enhancement would be worthy of study to test whether strong stretching can lead to midpoint breakage of molecules.