Holmes D L, Stellwagen N C
Department of Biochemistry, University of Iowa, Iowa City 52242.
J Biomol Struct Dyn. 1989 Oct;7(2):311-27. doi: 10.1080/07391102.1989.10507774.
Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.
通过在琼脂糖凝固时对其施加电场来制备定向琼脂糖凝胶。之后立即将DNA样品加到凝胶上，并在恒定的单向电场中进行电泳。无论定向场是平行还是垂直于最终的电泳方向施加，线性和超螺旋DNA分子的迁移率相比于同时运行的未定向对照凝胶，要么更快（80%的情况），要么更慢（20%的情况）。定向凝胶中迁移率的差异（无论是更快还是更慢）通常随着DNA分子量的增加以及用于使琼脂糖基质定向的电压的增加而增大。在垂直定向的凝胶中，线性DNA片段在向凝胶一侧倾斜的泳道中移动；超螺旋DNA分子在直线泳道中移动。如果定向电压平行于电泳方向施加，线性和超螺旋DNA分子都在直线泳道中迁移。在由不同类型琼脂糖制成的凝胶中，使用各种琼脂糖浓度和两种不同的运行缓冲液，并且在凝胶中添加或不添加溴化乙锭的情况下都观察到了这些效应。如果先让琼脂糖凝固，然后在加入DNA样品并进行电泳之前对凝胶施加定向电场数小时，也会观察到类似的结果。结果表明，琼脂糖基质可以在电泳前以及可能在电泳过程中通过施加电场来定向，并且基质的定向会影响DNA分子的迁移率和迁移方向。在垂直定向凝胶中观察到的倾斜泳道表明，通过施加电场可以在基质中形成孔隙或通道。随着时间的推移，定向基质会变得随机化，因为24小时后，定向凝胶和未定向凝胶中的DNA片段以相同的速度在直线泳道中迁移。
