Period Times and Helix Alignment during the Cyclic Migration of DNA in Electrophoresis Gels Studied with Fluorescence Microscopy

Abstract

Fluorescence microscopy of YOYO-stained DNA molecules has been used to study the periodicity of the cyclic migration during constant field electrophoresis in agarose gels. The distribution of period times is asymmetric and wide, with a ratio of standard deviation to mean value of 0.50 for fields between 3 and 24 V/cm. The average period time decreases with the electric field strength, E, as E -1.2 , and increases with increasing molecular weight (38-740 kbp) and increasing gel concentration (0.5-2%). The period times for T2 DNA (164 kbp) are in good agreement with the time to the orientation undershoot, as measured in a separate linear dichroism study of YOYO-stained DNA undergoing ordinary zone electrophoresis (Carlsson et al., manuscript in preparation). This shows that linear dichroism can be used to quantify the period time of the cyclic motion and to investigate effects of changes in DNA or gel properties. The cyclic conformational changes in the global DNA conformation during migration have been characterized in terms of the apparent length L μ and end-to-end distance h μ of the molecular paths in the gel. The formation of U conformations dominates the cycle time, since it is slow compared to U slipping and retraction. The maximum and cycle-averaged orientation factors for the apparent molecular path are evaluated from the images to be 0.70 and 0.67 ± 0.03, respectively, for T2 DNA at 8 V/cm in 1% agarose. From these values the degree of helix orientation below the level of microscope resolution could be estimated by comparison with the total orientation factor for the DNA helix as measured with linear dichroism. The maximum and cycle-averaged orientation factor for the helix with respect to the global path were 0.27 and 0.22 ± 0.01, respectively. These orientation factor values demonstrate (1) a very high degree of field alignment of the global path and (2) a degree of stretching of the helix along that path which is considerable, and stronger than expected for a wormlike chain spanning a cavity with the average pore size. If the imaged path corresponds to the reptation tube, the estimated friction coefficient for motion along the tube is found to be close to the value in the buffer.