Purification of DNA

Two general types of procedure are used for purification of DNA, centrifugation and chemical extraction. The principle of centrifugation is as follows. The sample is spun at high speed and the centrifugal force causes the larger or heavier components to sediment to the bottom of the tube. For example, destroying the cell wall of bacteria by lysozyme and detergents leaves a solution containing the fragments of the cell wall, which are small, and the DNA, which is a gigantic molecule. When the sample is cen-trifuged, DNA and some other large components are sedimented to the bottom of the tube. The fragments of cell wall, together with many other soluble components, remain in solution and are discarded.

The sedimented DNA is then re-dissolved in an appropriate buffer solution. However, it still has a lot of protein and RNA mixed in with it. These are generally removed by chemical means. One step used in many DNA purifications is phenol extraction. Phenol, also known as carbolic acid, is very corrosive and extremely dangerous because it dissolves and denatures the proteins that make up 60 to 70 percent of all living matter. Consequently, phenol may be used to dissolve and remove all of the proteins from a sample of DNA.

centrifugation Process in which samples are spun at high speed and the centrifugal force causes the larger or heavier components to sediment to the bottom detergent Molecule that is hydrophobic at one end and highly hydrophilic at the other and which is used to dissolve lipids or grease EDTA (ethylene diamine tetraacetate) A widely used chelating agent that binds di-positive ions such as Ca2+ and Mg2+ lysozyme An enzyme found in many bodily fluids that degrades the peptidoglycan of bacterial cell walls peptidoglycan Mixed polymer of carbohydrate and amino acids that comprises the structural layer of bacterial cell walls phenol extraction Technique for removing protein from nucleic acids by dissolving the protein in phenol

Add phenol

DNA, RNA plus protein in water layer c

Add phenol

DNA, RNA plus protein in water layer

Phenol layer

Phenol layer c

Shake!

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