Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

Abstract

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increases more than 10-fold after approximately 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change in the relaxation times of the orienting particles, the large increase in the amplitude of the birefringence must be due to an increase in the number of agarose fibers and/or fiber bundles orienting in the electric field, which in turn indicates a cooperative breakdown of the noncovalent "junction zones" that cross-link the fibers into the gel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the electric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly charged agarose), beta-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically cross-linked polymer) were also studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose gels in reversing fields is very similar to that of LE agarose gels, suggesting that the orientation anomalies are not due to the occasional charged residues on the agarose backbone chain. The beta-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agarose gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

“…The discordant agarose and polyacrylamide gel results are most likely due to the fact that the agarose gel fibers, which aggregate into large fiber bundles held together by hydrogen bonds [35], can be oriented by electric fields of the amplitudes typically used for agarose gel electrophoresis [35][36][37][38][39]. The orientation of the agarose fibers and fiber bundles effectively creates transient pores or "tunnels" through the gel matrix in the direction of the electric field [36].…”

Sequence‐dependent bending in plasmid pUC19

“…1A). Mobility anomalies are not observed for bent or curved molecules in agarose gels [12,26,27], in part because of the relatively large pores in agarose gels of the concentrations usually used for DNA gel electrophoresis [28,29], and in part because the agarose gel fibers tend to be oriented by the applied electric field [30][31][32], which would widen the pores further. By contrast, the permuted sequence isomers of pUC19 migrate with variable mobilities in large-pore polyacrylamide gels (Fig.…”

Effect of the matrix on DNA electrophoretic mobility

“…Agarose gel fibers have permanent dipole moments [54] and therefore interact with the electric field [54]; orientation occurs because the gel fibers are held together only by noncovalent hydrogen bonds [54,55]. The macroscopic orientation of the agarose gel fibers has been visualized directly by pre-electrophoresing an agarose gel in the direction perpendicular to the eventual direction of electrophoresis [56].…”