Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

Abstract

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 -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.Gel electrophoresis is the method of choice for the size fractionation of DNA in analytical biochemistry. Steady-field electrophoresis is commonly used to separate molecules from a few bp to about 20 kbp, while pulsed-field gel electrophoresis (PFGE) methods (1-3) are required to separate molecules beyond this size range, up to 10 megabase pairs (Mbp). In PFGE, the electric field is periodically alternated in two directions and DNA separation depends on the way the molecules reorient through the gel in response to the changing electric field (4, 5). Unfortunately, molecules longer than 1-2 Mbp can become permanently immobilized or trapped after traveling various distances through the gel, leading to band smearing (6). Trapping occurs in both steady-field and pulsedfield experiments if the electric field is higher than some critical value, E crit (7). The value of E crit falls as the DNA size is increased. Lowering the field below 2 V͞cm prevents trapping (6) but at the expense of very long electrophoretic runs (4). E crit appears to be higher for PFGE than for steady-field electrophoresis. Trapping in PFGE has been reduced, and sharper bands obtained by interrupting the long electric-field pulses with short high-voltage spikes in the reverse direction (7). These protocols notwithstanding, DNA trapping still remains a practical barrier to high-resolution PFGE above 10 Mbp. Understanding and preventing molecular trapping is essential to overcoming the current size limit of DNA separation and may lead to sharper resolution and reduced running times.Several mechanisms of DNA trapping have been proposed. Olson (6) suggested that very large molecules pile up against pores in a compressed state. Turmel and coworkers (7) suggest such pileups may occur in agarose cul-de-sacs because of locally high gel concentrations. Alternately, multiple ''hernias'' or ''impaled spirals'' (large DNA knots) could be the cause (7).Deutsch (8) has proposed that the molecules may bend around gel fibers with the two arms extending in the direction of the electric field, forming U-shapes. Because of local corrugation in both the DNA an...

Electrophoretic capture of circular DNA in gels

“…This observation showed that the trapping is reversible which is an important result since it is known that (linear) DNA can become trapped irreversibly at least in agarose gels [30], perhaps by a mechanism involving nicks in the DNA [31]. Reversible trapping of linear DNA has been observed as well, but it only occurs for DNA sizes above about 600 kbp [32].…”

Direct force measurements on DNA in a solid-state nanopore

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