“Flip‐flop” orientation of agarose gel fibers in pulsed alternating electric fields

The orientation of the agarose gel matrix in pulsed electric fields has been studied by transient electric birefringence. Two types of agarose with different degrees of charge were studied, in addition to agarose solutions and gels containing β-carrageenan, a stereoisomer of agarose, and polyacrylamide. Agarose gels exhibit normal orientation behavior when short, high voltage pulses are applied to the gel. The sign of the birefringence is positive and the relaxation times are consistent with the orientation of dangling fiber ends parallel to the electric field. When long, low voltage pulses, of the amplitude and duration used for pulsed field gel electrophoresis, are applied to the gel, completely different orientation effects are observed. The amplitude of the birefringence (i.e., extent of orientation) is much larger than expected from the high field results, and the birefringence decay curves contain multiple components of opposite sign. The relaxation times are consistent with the orientation of long agarose chain bundles or fibers, as well as large three-dimensional domains. Chain bundles or fibers of the lengths observed in the agarose gels are also observed in agarose solutions, suggesting that the fibers that are free to orient in 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 reverses from positive to negative in phase with the reversing electric field. This alternating change in the sign of the birefringence suggests 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 densities. The flip-flop orientation and reorientation of agarose fibers within the matrix in reversing electric fields may decrease the microscopic viscosity of the gel, increasing the mobility of large DNA molecules migrating through the gel during electrophoresis. Polyacrylamide gels do not exhibit an anomalous reversal of the sign of the birefringence in reversing electric fields. Hence, the orienting fibers in these gels do not change their direction of orientation in reversing electric fields. Extensive orientation is observed in β-carrageenan gels, similar to that observed in agarose gels. However, little orientation occurs in polyacrylamide gels, which are chemically crosslinked. Orientation of the agarose matrix also affects the mobilities observed for DNA restriction fragments during gel electrophoresis. If an agarose gel is oriented by applying an electric field to the molten agarose while it is solidifying, the mobilities of DNA fragments in the oriented gel differ by ∼10% from those observed in unoriented, control gels run at the same time. In addition, the trajectories of the migrating DNA molecules depend on the direction of the electric field used to orient the gel. The results suggest that orientation of the agarose gel matrix creates pores or channels in the direction of the applied electric field.