Separation of branched from linear DNA by two-dimensional gel electrophoresis

A general method for separating branched DNA molecules, such as replication forks and recombination intermediates, from linear forms has been developed. Using as a model a stable X-shaped molecule constructed in vitro, it was found that this branched form migrated more slowly during agarose gel electrophoresis than did a linear form of the same mass. Higher agarose concentrations and higher electrophoretic voltages enhanced the extent of retardation. These properties provided the basis for an electrophoretic method of separating branched from linear molecules by variation of agarose concentration and voltage over two dimensions. In the first dimension, concentration and voltage were low; in the second, both parameters were increased, thereby forcing X-shaped molecules to migrate to positions distinct from a diagonal arc of linear molecules. In addition, two-dimensional electrophoresis was capable of separating X-shaped forms of different mass from each other, as well as from linear molecules.