We will continue development of ultra-fast size measurements of unseparated nucleic acid fragment mixtures from restriction enzyme digests. We will measure electric birefringence relaxation frequencies using the frequency dispersion in agarose gels. We will develop protocols to reduce measurement time to less than 4 minutes. Heterodyning will be used to extend the upper frequency limit to 1 MHz. We will refine the theory of phase resolved frequency dispersion measurements to determine the ultimate resolution of the technique and to clarify problems in observed band shapes. Electric birefrigence will be used as a form of stainless on-line imaging densitometry during agarose gel electrophoresis of nucleic acids. On- line measurements will be used to control field inversions and field gradients and to minimize total running time. Instruments based on charge-coupled device cameras and linear diode arrays will be developed. Velocity modulation (AC+DC driving voltage) will be used to improve the sensitivity of capillary electrophoretic separations of proteins, peptides and nucleotides. Electrophoresis will be carried out with a high voltage DC power supply with a superimposed high voltage AC modulation signal. The modulation will generate a derivative (concentration gradient) signal with refractive index and other detectors. The modulated signal will be insensitive to heating, capillary movement and certain detector response fluctuations. Greatly improved detection limits are expected.
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