Isolation of circular DNA by zonal centrifugation: Separation of normal length, double length and catenated M13 replicative form DNA and of host specific “episomal” DNA

Abstract

The technique of zonal ultracentrifugation has been applied to the isolation and purification of large quantities of circular DNA. Using alkaline sucrose gradients several circular DNA species of different sizes could be isolated in a single purification step. 1. 1. M13 replicative-form DNA has been isolated on a large scale from infected cells. Analysis in the analytical ultracentrifuge showed that the infective replicative form was native, sedimenting with 24 S. 2. 2. In addition to the normal-length replicative form of M13, double-length and catenated forms occurring in small amounts in infected cells were isolated. Evidence for this is provided by measurement of the contour length and by test for infectivity. 3. 3. From infected and uninfected cells, large circular DNA molecules were isolated with a contour length of 34 μ. The molecular weight was estimated to be about 65·10 6 , and it is suggested that these molecules represent bacterial episomes whose size approximates that of F factors.