Preparative separation of DNA--ethidium bromide complexes by zonal density gradient centrifugation.

Restriction enzyme fragmentation and recombinant DNA technology offer powerful tools with which to study gene structure, organization, and expression. Frequently in these studies the need arises to purify on a preparative scale a specific DNA molecule from a mixture. The two most commonly used procedures in such purification are isopycnic centrifugation in dye-salt gradients and gel electrophoresis. Dye-buoyant density centrifugation allows excellent separation and purification of covalently closed circular (CCC) DNA molecules from DNA of other conformations (l-2). However, the procedure does not permit the separation of different kinds of DNA molecules if they have a similar conformation. Electrophoresis through polyacrylamide or agarose gels separates nucleic acids primarily on the basis of size and conformational difference (3-5). However, any DNA passed through a gel is contaminated with fragments of the gel matrix. The presence of gel material often interferes with the further