The design of effective separation systems for bent and other forms of conformationally """"""""abnormal"""""""" DNA requires an understanding of the causes of gel retardation. We have sought such understanding by computer simulations of the passage of variously shaped objects through arrays of obstacles, i.e. a highly simplified model of a gel. We have also initiated a study in polymer solution - and in """"""""Mutation Detection"""""""" - electrophoresis (MDE) to correlate polymer structure with retardation. 1) Computer modeling of the passage of variously shaped particles through arrays of circular obstacles demonstrated progressive retardation of rod-shaped particles with bending; a rate of passage of circular particles greater than that of rods at low densities of obstacles and less than that of rods at higher densities, with complete arrest at a sufficient obstacle density; progressive rates of passage upon increase in flexibility of the particle; decreasing rates of passage with increasing axial ratio or curvature and, most dramatically, increasing length of curved surface. These rates of passage correlate with the mobilities of the analogous DNA conformers. 2) Using the same technique of computer modeling, the applicability of the Ogston model to prolate ellipsoid particles was demonstrated. Application to non-spherical particles requires prefactors which were defined for prolate ellipsoids and can potentially be defined for other non-spherical particle shapes. 3) DNA electrophoresis conducted on agarose gels submerged under an immiscible solvent followed by ethidium bromide staining provides relative fluorescence intensities that are independent of DNA size in the range investigated (up to 2 kb) while those derived from the conventional """"""""submarine"""""""" technique fall off sharply with DNA size. 4) The separation of standard from heteroduplex DNA on a transverse MDE gel is independent of gel concentration. The finding allows for shortened MDE gel analysis, and suggests a pseudo-charge separation mechanism for """"""""kinked"""""""" DNA. 5) Intrinsic viscosity values and retardation coefficients for polystyrene sulfate size standards were determined in solutions of polyacrylamide dextran, polyvinyl alcohol, polyethylene glycol, hydroxyethyI cellulose and polyvinylpyrrolidone of various sizes. An attempt is being made to correlate particle size and effective polymer size for separation within a given particle size range. These combined studies have shown that a) the """"""""abnormal"""""""" retardation of bent and circular small DNA is likely to be purely due to geometric properties and is accentuated through molecular flexibility. b) Delicate conformational changes which are not expressed by the shape of the electrophoretic pattern in a gel concentration gradient (""""""""kinks"""""""") are responsible for resolution in MDE. c) Molecular sieving of large macromolecules seem most effective at low polymer concentrations and commensurate end-to-end distances of polymers in solution. These findings provide elements for the rational design of polymer architecture for effective molecular sieving.
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