Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels
Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels
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DOI：
10.1016/s0006-3495(98)78020-8
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发表时间：
1998-06-01
影响因子：
3.4
通讯作者：
Åkerman, B
中科院分区：
文献类型：
--
作者：
Åkerman, B
电泳速度和方向已被用来研究电场诱导的捕获超螺旋和松弛的环状DNA（2926和5386 bp）在聚丙烯酰胺凝胶（5%T，3.3%C）在7.5-22.5 V/cm，作为控制的线性分子的轮廓长度或相同的回转半径。环特异性捕获是可逆的。从所需的反向脉冲的持续时间去陷阱的分子，平均陷阱深度估计为90，这是一致的分子电荷和所需的场强，以保持分子被困。陷获的圆表现出较强的场对准相比，线性形式，并有一个很好的相关性之间的增强场对准的圆和发病的捕获在恒定和脉冲字段。圆形没有表现出对线性DNA所见的场脉冲的取向过冲响应，并且取向生长的速率随场缩放为E-2+/-0.1，与线性形式的E-1.1+/-0.1相反。这些结果表明，线性形式迁移的循环爬行，而圈最有可能被困在凝胶纤维刺穿。这一提议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，其中由于更硬的凝胶纤维，刺穿被认为更有可能。捕获效率对DNA拓扑结构敏感，正如刺穿所预期的那样。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物质低2 - 4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plectonemic超螺旋结构中的平均孔和两种凝胶类型中的纤维厚度的现有数据一致。拓扑结构效应的基础上，有人认为，在聚丙烯酰胺凝胶脉冲场电泳过程中的穿刺可能是有用的分离更复杂的DNA结构，如结。结果还表明，线性二色性场取向的分子可以用来测量超螺旋角，如果放松的DNA圈被用作控制的全球程度的取向。
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.