Trapping of megabase-sized DNA molecules during agarose gel electrophoresis.
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 lambda-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.
Gurrieri
S
S
Dipartimento di Scienze Chimiche, Universitá di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Smith
S B
SB
Bustamante
C
C
D010582:Bacteriophage lambda / Q000235:genetics; D002875:Chromosomes / Q000378:metabolism; D004271:DNA, Fungal / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D004279:DNA, Viral