Propidium diiodide-cesium chloride density gradient centrifugation for buoyant separations of duplex DNA molecules containing single-stranded regions.
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.
D002499:Centrifugation, Density Gradient / Q000379:methods; D002586:Cesium; D003090:Coliphages; D004270:DNA, Circular / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification*; D004926:Escherichia coli; D007202:Indicators and Reagents
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D007202:Indicators and Reagents; D002586:Cesium