Isolation of Bacterial DNA

To isolate a functional macromolecular component from bacterial cells, you must accomplish three things. First, you must efficiently disrupt the bacterial cell wall and cell-membrane system to facilitate extraction of desired components. Second, you must work under conditions that either inhibit or destroy the many degradative enzymes (nucleases, proteases) released during cell disruption. Finally, you must employ a fractionation procedure that separates the desired macromolecule from other cellular components in satisfactory yield and purity. The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium dodecyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. This experiment employs a variety of fractionation methods to purify the bacterial DNA. Perchlorate ion is used to dissociate proteins from DNA. Chloroform–isoamyl alcohol is used to denature and precipitate proteins by lowering the dielectric constant of the aqueous medium. The precipitated material is removed by centrifugation. DNA is then isolated from the solution by ethanol precipitation, which allows spooling of the long, fibrous DNA strands onto a glass rod. Although the residual RNA and proteins in the solution also coagulate, they do not form fibers and are left in the solution as a granular precipitate. The resultant crude DNA is dissolved and precipitated again with isopropanol under conditions that favor specific DNA precipitation. These steps form a general procedure for the isolation of chromosomal DNA from virtually any bacterium. If you intend subsequently to assay the biological activity of the isolated DNA in a transformation assay (as in Experiment 20), it is necessary to start this isolation with the appropriate species and strain of bacteria possessing the genotype of interest. This experiment will also provide you with experience in characterizing DNA samples by determining their ultraviolet absorption spectra and observing their thermal denaturation by means of the hyperchromic effect. These topics were discussed in the introduction to Section V.