The use of transient electric birefringence to characterize the conformation of DNA in solution, the mechanism of DNA gel electrophoresis, and the structure of agarose gels

Abstract

Abstract Two small, 153 base-pair (bp) DNA restriction fragments, one of which migrates anomalously slowly during electrophoresis in polyacrylamide gels, have been characterized by transient electric birefringence (TEB). The decay of the birefringence of the anomalously slowly migrating fragment is faster than that of the normal fragment under a variety of buffer conditions, suggesting that the anomalous fragment is stably bent or curved in free solution. Reversing field experiments suggest that both the anomalous and normal DNA fragments orient in the electric field by a slow induced dipole mechanism. Birefringence relaxation times have also been measured for three DNA molecules embedded in agarose gels of various concentrations. The embedded DNA molecules become oriented in the electric field, just as in free solution. If the median pore diameter of the gel is approximately twice as large as the apparent end-to-end length of the DNA in solution, the relaxation times of the embedded DNA molecules are unaffected by the presence of the gel, indicating that the DNA retains its usual wormlike coil conformation in the gel matrix. However, as the end-to-end length of the DNA approaches the median pore diameter of the gel, the DNA molecules become stretched out and elongated in the electric field, leading to a repetitive, or end-on, mode of migration during electrophoresis. The relaxation times of the stretched DNA molecules scale as the 2.8 power of molecular weight, in reasonable agreement with reptation theories. The structure of agarose gels has also been characterized by TEB. The long, low voltage electric fields used for pulsed field gel electrophoresis (PFGE) cause extensive orientation of the agarose gel fibers. A wide range of relaxation times is observed after the field is removed, corresponding to the orientation of gel fibers up to 22 μm in length. The sign of the birefringence is variable, indicating that the gel fibers can be oriented in different directions in different parts of the gel. Surprisingly, the sign of the birefringence reverses upon field reversal, suggesting that the gel fibers change their direction of orientation in reversing electric fields. This anomalous orientation behavior is not observed for chemically crosslinked polyacrylamide gels. The apparent orientation and reorientation of agarose gel fibers in rapidly reversing electric fields may lead to ‘dynamic pores’ in the gel matrix, facilitating the migration of very large DNA molecules during PFGE.