The project will investigate fundamental phenomena in electrophoresis gels and apply these results to novel forms of gel electrophoresis and detection of nucleic acids and proteins in gels. Both slab gel and capillary formats will be investigated. The dynamics of gel compression and extension will be investigated using electric birefringence and fluorescence polarization imaging of slab gels and gel-filled capillaries. The effects of dynamic gel distortion on electrophoretic mobilities of nucleic acids will be investigated, and used to propose an improved separation model. Electric birefringence will be used for stainless and non-nicking imaging of agarose and polyacrylamide electrophoresis gels. Numerical techniques will be used to remove artifacts and background from the images to achieve subnanogram sensitivity. On-line imaging will be used for real-time observation and to control pulse parameters in several forms of pulse-field electrophoresis, with the goal of optimizing separations and minimizing running times. Electric birefringence will also be used as a detector principle for nucleic acids in pulsed field capillary gel electrophoresis. Diode laser excitation and lock-in amplifier extraction of birefringence will be used to achieve high detection sensitivity. Pulsed field capillary gel electrophoresis will be developed. In conventional quartz capillaries, the effect of different pulsing regimes will be studied. Agarose-loaded cellulose acetate capillaries will be used with external electrodes to implement orthogonal field capillary gel electrophoresis and related techniques, which are expected to extend the utility of CGE to the 100 kbp size range or higher. Raman microspectrometry will be used to investigate structural changes in polyacrylamide and other gels in capillaries, to determine the factors governing capillary life times.
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