ELECTRIC BIREFRINGENCE OF DNA RESTRICTION FRAGMENTS

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The research described in this proposal is designed to investigate the molecular basis of the anomalously slow electrophoretic mobilities observed for certain DNA fragments in polyacrylamide gels, investigate the internal structure of the agarose gel matrix and the orientation of agarose gels in pulsed electric fields, and characterize the transverse induced dipole moment of DNA molecules in solution. Kilobase-sized DNA molecules with permuted sequences will be studied by the circular permutation assay and by transient electric birefringence, to see whether their gel mobilities correlate with their apparent end-to-end lengths. Anomalously slowly migrating restriction fragments located at the apparent bend centers of the DNA molecules, and midway between the apparent bend centers, will also be studied, to determine whether structural differences exist between these two classes of anomalously slowly migrating restriction fragments. The mechanism of orientation of DNA molecules in an electric field will be studied by electric birefringence, to see whether the initial direction of orientation is determined by a fast transverse induced dipole moment. The electric birefringence of agarose gels will be determined at various locations within each gel. The intrinsic (gelation induced) birefringence of the agarose gel matrix will also be measured before and after applying electric field pulses to the gel. The long term goals of this research are to characterize the sequence- dependent conformational variability of DNA, to understand the molecular basis of DNA gel electrophoresis, and to characterize the mechanism of orientation of DNA in an electric field.