ELECTRIC BIREFRINGENCE OF DNA RESTRICTION FRAGMENTS

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The interaction of DNA restriction fragments with electric fields will be investigated using the technique of transient electric birefringence. Particular emphasis will be placed on a study of the orientation and disorientation of DNA restriction fragments imbedded in agarose and in polyacrylamide gels. Electric birefringence is a particularly useful technique for these studies, because it measures directly the orientation of the DNA molecules in the gel matrix. Orientation will be studied with pulsed electric fields of different amplitudes and durations, with a variable delay between pulses, and with rapidly reversing electric fields. Orientation in a solution containing linear polyacrylamide molecules of different charge densities will also be studied. Electric birefringence studies will be continued on the orientation, disorientation, reversing field, and saturation behavior of two 147 base pair restriction fragments obtained from the plasmid pBR322. These two fragments differ in their mobility on polyacrylamide gels, and in other physical properties, and typify sequence-related structural differences in the Watson- Crick helix. Other pairs of normally and abnormally slowly migrating DNA fragments will be studied, as well as synthetic polynucleotides. Additional electric birefringence studies will focus on the saturation and reversing field behavior of DNA fragments of different molecular weights in free solution, the B A conformational transition of DNA restriction fragments, the binding of restriction enzymes to DNA fragments, the binding of ethidium bromide and other ligands to DNA fragments, the binding of ethidium bromide and other ligands to DNA fragments, and the electric birefringence of linear polyacrylamides of different molecular weights and charge densities. Many of the electric birefringence measurements will be correlated with circular dichroism and/or gel electrophoretic studies. The result of these experiments will clarify the mechanism of gel electrophoresis in different gel media and identify some of the effects of sequence-dependent conformation differences on the physical properties of DNA. The long range goals of this research are to understand the behavior of DNA molecule in solution and to characterize the mechanism of interaction of DNA and other polyions with electric fields.