The polysacharide gellan forms hydrogels if a divalent ion such as calcium is added at millimolar concentrations. The gel can be reversed to solution by adding EDTA, which makes it a promising candidate for preparative electrophoretic separation of biomolecules. We have studied the electrophoretic migration of double-stranded T4 DNA (164 kilobasepairs) in gellan gels by velocity measurements and linear dichroism spectroscopy studies of the DNA coil conformation during the migration. The gels either contained 0.3 % of high molecular weight (5 106) poly(ethylene oxide) (PEO), in order to suppress the electroosmosis induced by the negative charges of the gellan polymer, or were free of such added polymer and therefore exhibited an electroosmotic flow which is opposite to the DNA migration. In both cases does the viral DNA migrate in an oscillatory manner between stretched and coiled states, because it becomes entangled with the gellan gel fibers. In the stretched state of the cycle the molecules are substantially aligned with the field. As the field is turned off the alignment relaxes first by a rapid (seconds) destretching along the path in the gel, followed by a slower (minutes) end-on type of motion to the equilibrium isotropic coil state. The well-understood migration and relaxation behavior was exploited to investigate the effect of the electroosmotic flow on the DNA migration, and the effect of the PEO added to quench this retarding flow. The electroosmotic flow strongly reduces the electrophoretic stretching of the DNA, but has small effects on the path chosen by the DNA through the gel. The added PEO has two effects on the DNA migration. An indirect effect is that the quenched electroosmosis leads to a stronger stretching of the DNA, and to shorter cycle period times because the ends of the molecules move faster in the absence of the counterflow. A direct effect is that the PEO itself retards the DNA motion, most likely due to a combination of hydrodynamic interactions and entanglement effects. The net result of these two opposing PEO-effects is that the center-of-mass velocity increases by a factor of about 2 upon addition of PEO. Circular plasmid DNA(13 kbp) can be electrophoretically trapped in gellan gels containing PEO.
Citation: Journal of Physical Chemistry B
Issue: No. 9
Pub Type: Journals
agarose, DNA, electroosmosis, electrophoresis, gel, gellan, plasmid, separation, trapping