Electrostatic confinement and manipulation of DNA molecules for genome analysis

Repeated sequences make up approximately two-thirds of the human genome, which become fully accountable when very large DNA molecules are analyzed. Long, single DNA molecules are problematic using common experimental techniques and fluidic devices because of mechanical considerations that include breakage, dealing with the massive size of these coils, or the huge length of stretched DNAs. Accordingly, a team of researchers at the University of Chicago’s IME and the University of Wisconsin – Madison has found a way of harnessing analyte “issues” as exploitable advantages by invention and characterization of the “molecular gate,” which controls and synchronizes formation of stretched molecules as DNA dumbbells within nanoslit geometries that may also offer new routes to separation. This was accomplished by theoretical studies and experiments leveraging a series of electrical forces acting on DNA molecules, device walls, and the fluid flows within our devices.

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways that, as shown by IME and Wisconsin researchers, effectively create an “electrostatic bottle.” This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which guided the team’s efforts to optimize design and functionalities. In addition, it was demonstrated that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasma florum genome, via synchronized dumbbell formation, was used to validate the team’s nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

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