Transient electric birefringence of agarose gels. I. Unidirectional electric fields.

Abstract

The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N.C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537-1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to approximately 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to approximately 22 microns in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Kerr constants are approximately 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increases approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields. The optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE.