The Green Phoenix: A History of Genetically Modified Plants

By Paul F. Lurquin | Go to book overview

APPENDIX 1
Cesium Chloride Density Gradients
and Their Use in DNA Analysis

Cesium chloride (CsCl) is a very dense salt. It is also extremely soluble in water, and highly concentrated solutions (e.g., 6 mol/L) are used to run density gradients. The technique of CsCl equilibrium density gradient centrifugation for DNA analysis was invented in 1957 by Meselson and Stahl. The underlying idea is that Cs+ ions (always neutralized by Cl_ ions) will sediment to the bottom of a tube when subjected to high g-forces. These forces can be achieved if the tube is spun in a centrifuge rotor at speeds of at least 30, 000 rpm. The sedimentation of the Cs+ ions is constantly counteracted by thermal diffusion, which forces these ions to move randomly in the tube. After enough time (typically 60 hours, or less, depending on the g-force applied, which depends on rotor speed and size), an equilibrium is established between sedimentation and thermal diffusion; a gradient thus forms in the tube, in which the concentration of Cs+ is highest at the bottom of the tube and lowest at the top. This gradient is perfectly linear. DNA from all organisms has a density of about 1.7. Further, this density is directly dependent on the DNA (G + C) content, which has a characteristic value for each organism but differs among diverse kinds of plants animals, bacteria, and viruses. The higher the G + C content, the higher the density. Therefore, DNA molecules with different G + C contents will band (literally float) in the gradient at a place where they encounter their own density, at a certain CsCl concentration. They cannot sediment farther because a higher CsCl concentration (a higher density) will push them up; thermal diffusion, which would randomize the movement of these molecules, is exactly counteracted by g-forces, which push them down. This makes DNA bands quite narrow, and DNAs with different G + C contents

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