Trapping of megabase-sized DNA molecules during agarose gel electrophoresis
Trapping of megabase-sized DNA molecules during agarose gel electrophoresis
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DOI：
10.1073/pnas.96.2.453
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发表时间：
1999-01-19
影响因子：
11.1
通讯作者：
Bustamante, C
中科院分区：
文献类型：
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作者：
Gurrieri, S;Smith, SB;Bustamante, C
在电泳过程中，如果电场超过每厘米几伏，大分子DNA分子就会被困在琼脂糖凝胶中。荧光显微镜显示，这些分子总是以U形构象停滞。现场vs.捕获的尺寸依赖性表明捕获需要临界分子张力。在给定场的凝胶电泳过程中剪切的未连接的h-梯的尺寸与在该场捕获的分子的尺寸一致，这表明两个过程都是通过U形顶点附近的切口熔化发生的。一致地，通过暴露于紫外线辐射而产生缺口的分子比未暴露的分子更容易捕获。在顶点处的临界捕获张力估计为15 pN，该力足以熔化围绕凝胶纤维弯曲的切口，并且根据我们的模型，捕获分子。降低分子张力和避免捕获的策略进行了讨论。
Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm, Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping, The size of unligated h-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.