The overarching goal of the project is development of high speed, high resolution electrophoretic separations based on pulsed field capillary gel electrophoresis in ultradilute sieving buffer. In pursuit of that goal we will undertake a wide-ranging study of the mechanism of electrophoretic separation in ultradilute sieving buffers, using fluorescence microscopy and electrophoretic separations as the main tools. By fluorescence microscopy we will investigate whether simple mechanical collision or polymer entanglement is necessary for topology changes in DNA. Derivatized celluloses and polyethylene oxide will be the entangling polymers. The stoichiometry, lifetime and relaxation dynamics of representative adducts will be measured. This information will be used to test correlation with efficient (resolution-enhancing) field inversion electrophoresis protocols. Microscopy will be used to investigate the transition between the ultradilute solution regime and the polymer entanglement regime and to ascertain whether certain polymers or pulse protocols promote the existence of multiple stable topologies for DNA, thus leading to band splitting. We will then develop pulsed field capillary gel electrophoresis for nucleic acid separations in the size range 100 kbp- 10 Mbp, using ultradilute solutions of polyethylene oxide and other ultralong chain polymers and will optimize these using simplexes or other experimental design strategies. If the mechanical interaction hypothesis is proved valid, we will attempt electrophoretic separations of 10-50 Mbp DNA using microspheres instead of polymer chains. Semi-preparative versions of the most efficient separation protocols will be developed. DC and pulsed field capillary electrophoresis in ultradilute solutions will be extended to polysaccharides. The target systems will be heparins for DC electrophoresis and dextrans for pulsed field electrophoresis.
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