Propidium diiodide-cesium chloride density gradient centrifugation for buoyant separations of duplex DNA molecules...

At increasing dye concentrations in propidium diiodide-CsCl density gradients, the relative buoyant density shift was largest for open-circular duplex phiX174 DNA (RFII), next largest for single-stranded viral DNA, and least for closed-circular duplex DNA (RFI). These differential relative buoyant density shifts permitted discrete separations of these phiX DNA forms. Further, in propidium diiodide-CsCl density gradients, the distinctive density of single-stranded DNA permitted separations of rolling-circle intermediates with a single-stranded tail that occur during single-stranded phiX DNA synthesis. It is suggested that single-strandedness in a duplex DNA structure influences its buoyand density shift due to differential dye binding to the single-stranded region and is an additional physical basis for relative buoyant separations of DNA molecules in propidium diiodide-CsCl density gradients.