284777 DNA Gel Electrophoresis in the Entropic Trapping Regime: A Versatile Tool for Enhanced Separations and Nanostructural Analysis
Macromolecules confined within nanoporous surroundings experience entropic trapping (ET) when their dimensions approach the average pore size, leading to emergence of transport behavior that can be immensely beneficial (e.g., a counterintuitive trend of increasing separation efficiency with DNA size during gel electrophoresis). But the noisy uncorrelated process by which the embedded macromolecules discretely hop from pore to pore contributes additional dispersion that detrimentally impacts most practical applications. Here we show how these limitations can be overcome by imposing an oscillatory electric field at a period tuned to the activation timescale of ET in ordinary hydrogels, establishing a resonance condition that synergistically combines accelerated mobility and reduced diffusion. This effect can be exploited to induce bi-directional transport of different sized DNA fragments owing to the size-dependence of the optimal modulation period. We show this in microchip electrophoresis by achieving a state in which neighboring bands in the gel travel in opposite directions.
We also show how this resonance phenomenon can be harnessed as a sensitive probe of DNA binding interactions by exploiting the fact that the conditions under which resonance emerges strongly depend on the DNA coil size. Therefore, binding interactions become visible as a distinct peak in electrophoretic mobility occurring at a conformation-dependent electric field actuation period. These measurements also enable structural information (persistence and contour lengths) to be extracted by correlating the period at maximum mobility with conformational characteristics of the DNA complex. There is no inherent lower limit on the DNA size that can be interrogated, as opposed to single-molecule studies which require large DNA (λ-phage or longer).
See more of this Group/Topical: Topical 3: 2012 Annual Meeting of the American Electrophoresis Society (AES)