Isopycnic centrifugation

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Isopycnic centrifugation or equilibrium centrifugation is a process used to isolate nucleic acids such as DNA. To begin the analysis a mixture of cesium chloride and DNA is placed in a centrifuge for several hours at high speed to generate a force of about 10^5 x g (earth's gravity). Cesium chloride is used because at a concentration of 1.6 to 1.8 g/mol it is similar to the density of DNA. After some time a gradient of the cesium ions is formed, caused by two opposing forces: diffusion and centrifugal force. The diffusive force arises due to the density gradient of solvated cesium chloride and it always directed towards the center of the rotor. The sedimenting particles will sediment away from the rotor until their density is equivalent to the local density of the cesium gradient, at which point the diffuseive force is equivalent to the centrifugal force. The DNA molecules will then be separated based primarily on the relative proportions of AT (adenine and thymine base pairs) to GC (guanine and cytosine base pairs). An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal.

Reference

• Gerald Karp, Cell and molecular biology: Concepts and experiments, fourth edition, 2005, Von Hoffman press

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