FLIP-FLOP ORIENTATION OF AGAROSE-GEL FIBERS IN PULSED ALTERNATING ELECTRIC-FIELDS

The orientation of the agarose gel matrix in pulsed electric fields ha s been studied by transient electric birefringence. Two types of agaro se with different degrees of charge were studied, in addition to agaro se solutions and gels containing beta-carrageenan, a stereoisomer of a garose, and polyacrylamide. Agarose gels exhibit normal orientation be havior when short, high voltage pulses are applied to the gel. The sig n of the birefringence is positive and the relaxation times are consis tent with the orientation of dangling fiber ends parallel to the elect ric field. When long, low voltage pulses, of the amplitude and duratio n used for pulsed field gel electrophoresis, are applied to the gel, c ompletely different orientation effects are observed. The amplitude of the birefringence (i.e., extent of orientation) is much larger than e xpected from the high field results, and the birefringence decay curve s contain multiple components of opposite sign. The relaxation times a re consistent with the orientation of long agarose chain bundles or fi bers, as well as large three-dimensional domains. Chain bundles or fib ers of the lengths observed in the agarose gels are also observed in a garose solutions, suggesting that the fibers that are free to orient i n the gels had previously formed in the sol phase and are only weakly integrated into the matrix structure. In rapidly reversing low voltage electric fields, the sign of the birefringence of the agarose gels re verses from positive to negative in phase with the reversing electric field. This alternating change in the sign of the birefringence sugges ts that the agarose fibers ''flip-flop'' in orientation from parallel to perpendicular every time the electric field reverses its direction. Similar effects are observed for agarose gels with different charge d ensities. The flip-flop orientation and reorientation of agarose fiber s within the matrix in reversing electric fields may decrease the micr oscopic viscosity of the gel, increasing the mobility of large DNA mol ecules migrating through the gel during electrophoresis. Polyacrylamid e gels do not exhibit an anomalous reversal of the sign of the birefri ngence in reversing electric fields. Hence, the orienting fibers in th ese gels do not change their direction of orientation in reversing ele ctric fields. Extensive orientation is observed in beta-carrageenan ge ls, similar to that observed in agarose gels. However, little orientat ion occurs in polyacrylamide gels, which are chemically crosslinked. O rientation of the agarose matrix also affects the mobilities observed for DNA restriction fragments during gel electrophoresis. If an agaros e gel is oriented by applying an electric field to the molten agarose while it is solidifying, the mobilities of DNA fragments in the orient ed gel differ by approximately 10% from those observed in unoriented, control gels run at the same time. In addition, the trajectories of th e migrating DNA molecules depend on the direction of the electric fiel d used to orient the gel. The results suggest that orientation of the agarose gel matrix creates pores or channels in the direction of the a pplied electric field.