Single molecule analysis of DNA electrophoresis in microdevices

Abstract

Given that current electrophoresis technology is inadequate for mapping large O[100 kilobasepair] DNA, several promising lab-on-chip designs for DNA mapping have been recently proposed that require either 1) a DNA molecule negotiating an obstacle course in a microchannel or 2) stretching a DNA molecule for linear analysis. The goal of this research is to experimentally probe the fundamental physics that underlie these DNA mapping designs. Furthermore, we present a continuous stretching process that offers significant improvement over results in literature. In general, the governing physics is complex due to the confinement of the microchannel, the coiled-nature of long DNA molecules, and the induced electric field gradients from obstacles and changes in channel dimensions. Using single DNA fluorescence microscopy, we have investigated many of the governing physical mechanisms at play in these gene mapping microfluidic devices. For example, we have determined transport coefficients for DNA in a confined channel and performed an analysis of hydrodynamic background flows in these thin channels. We have also performed a systematic study of the impact and unhooking dynamics of a DNA molecule driven into a small stationary post.

(cont.) Furthermore, we have thoroughly investigated DNA stretching in electric field gradients created by a contraction and a large insulating obstacle. Just as a flow gradient stretches a polymer, an electric field gradient can stretch a charged polyiner like DNA. Because electric field gradients have no local rotational components, a charged polymer will experience purely extensional deformation. This is true even near surfaces, unlike the cyclic extension-compression dynamics characteristic in shear flows. A recurring theme is this work is the notion of configuration effects, or "molecular individualism", a term coined by DeGennes eight years ago to denote the sensitive dependence of a single polymer's deformation dynamics to its initial configuration. New mapping designs often require or force DNA to strongly deform and we see molecular individualism arise when studying phenomena central to these mapping designs, e.g. DNA colliding into stationary posts or DNA stretching in electric field gradients. Molecular individualism counteracts mapping and other potential engineering applications that might require uniform behavior of all DNA molecules. However, our results show that molecular individualism can be bypassed by pre-configuring the DNA configurational distribution before strong deformation.

(cont.) The main impact of these findings will be to foster the design of custoil unit operations in biomolecule microfluidic processes, e.g. for DNA mapping applications and beyond.