Akerman B
Department of Physical Chemistry, Chalmers University of Technology, Göteborg, Sweden.
Biophys J. 1998 Jun;74(6):3140-51. doi: 10.1016/S0006-3495(98)78020-8.
Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.
利用电泳速度和取向研究了在7.5 - 22.5 V/cm电场作用下，超螺旋和松弛环状DNA（2926和5386 bp）在聚丙烯酰胺凝胶（5% T，3.3% C）中的捕获情况，使用相同轮廓长度或相同回转半径的线性分子作为对照。环状DNA特有的捕获是可逆的。根据使分子解捕获所需反向脉冲的持续时间，估计平均捕获深度为90 Å，这与分子电荷以及使分子保持捕获状态所需的场强一致。与线性形式相比，捕获的环状分子表现出强烈的场取向，并且在恒定场和脉冲场中，环状分子增强的场取向与捕获的开始之间存在良好的相关性。环状分子对场脉冲没有表现出线性DNA所具有的取向过冲响应，并且取向增长速率与场强的关系为E(-2±0.1)，而线性形式为E(-1.1±0.1)。这些结果表明，线性形式通过循环爬行迁移，而环状分子很可能是被凝胶纤维刺穿而捕获。这一观点得到了琼脂糖凝胶中环状DNA非常相似的速度和取向行为的支持，在琼脂糖凝胶中，由于凝胶纤维更硬，刺穿被认为更有可能发生。捕获效率对DNA拓扑结构敏感，这符合刺穿的预期。在聚丙烯酰胺中，超螺旋形式（超螺旋密度σ = -0.05）的捕获概率比相应的松弛形式低两到四倍，而在琼脂糖凝胶中，超螺旋形式根本不会被捕获。这些结果与关于螺旋超螺旋结构中平均孔洞以及两种凝胶类型中纤维厚度的现有数据一致。基于拓扑效应，有人认为在聚丙烯酰胺凝胶中进行脉冲场电泳时，刺穿可能有助于分离更复杂的DNA结构，如结。结果还表明，如果使用松弛的DNA环状分子作为整体取向程度的对照，对场取向分子的线性二色性可用于测量超螺旋角。
