The electric field dependence of DNA mobilities in agarose gels: A reinvestigation

Abstract

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments ≤ 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained ≤ 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris‐borate buffer (TBE) than in gels cast and run in Tris‐acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incroporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature‐corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments ≤ 12 kbp in size in agarose gels ≤ 1.4 % in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments ≤ 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments ≥ 1 kbp in size in agarose gels ≥ 2 % in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an “intrinsic” DNA mobility of 2.7 x 10⁻⁴ cm²/Vs at 20°C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments. If the volume occupied by each DNA molecule is approximated by its root‐mean‐square (rms) radius of gyration, the median pore radius, r_p, of an LE agarose gel cast and run in TAE buffer is r_p = 98 A^−0.90, where A is the agarose concentration in % and r_p is given in nm. For gels cast and run in TBE buffer, the apparent median pore radius is r_p = 66 A^−0.95. If the volume occupied by each DNA molecule is represented by its mean geometric radius instead of the rms radius of gyration, the median gel pore radius in both buffers depends on A^−0.5, as predicted by the extended Ogston theory. From the corresponding retardation coefficients, the gel fiber parameters were estimated.