Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

Megabase DNA molecules become trapped in agarose gels during electrophoresi s 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 molecu lar tension is required for trapping, The size of unligated h-ladders, shea red during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur thro ugh nick melting near the vertex of the U-shape. Consistently, molecules ni cked 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 mode l, trap a molecule. Strategies to reduce molecular tension and avoid trappi ng are discussed.