Section 1 update: Simplified protocols for the preparation of genomic DNA from bacterial cultures (original) (raw)

2008, Molecular Microbial Ecology Manual

in the handling of the preparations, which are necessary for obtaining genomic DNA of high molecular weight. Thus, in general, the most desirable means of disrupting bacterial cells for obtaining genomic DNA is through enzymatic digestion and detergent lysis. Such a strategy is enhanced by prior treatment of cells with a metal chelating agent, such as ethylenediamine-tetraacetic acid (EDTA). If the cell wall of the organism is susceptible to such treatments, relatively high molecular-weight genomic DNA can be obtained which is applicable for a number of analytical techniques. Further, the lysis should be carried out in a buffered (pH 8-9) medium containing EDTA. The alkaline pH reduces electrostatic interactions between DNA and basic proteins, assists in denaturing other cellular proteins and inhibits nuclease activities. EDTA binds divalent cations, particularly Mg 2+ and Mn 2+ , reducing the stabilities of the walls and membranes and also inhibits nucleases which have a requirement for metal cations. Cell disruption by enzymatic treatments Lysozyme, isolated commercially from chicken egg white, is a member of the broad class of muramidases which catalyse the hydrolysis of the β-1,4-glycosidic linkage between the N-acetylmuramic acid-N-acetylglucosamine repeating unit, comprising a major part of the peptidoglycan layer of the cell walls of most bacteria [18]. Lysozyme is especially effective in disrupting bacterial cells when used in combination with EDTA [15]. Lysozyme and related enyzmes are useful for disrupting the cells of a broad range of bacterial species, although many species are not particularly susceptible to muramidase treatment due, presumably, to layers of protein or capsular slime, which protect the peptidoglycan. Additionally, as their cell walls do not contain peptidoglycan, all described species of Archae are resistant to lysozyme activity. Proteinase K, a serine protease produced by the fungus Tritirachium album, cleaves adjacent to the carboxyl groups of aliphatic and aromatic amino acids involved in peptide bonding [4], including those comprising the peptide crosslinking interbridges of the peptidoglycan layers of the cell walls of bacteria. The applicability of Proteinase K for disrupting bacterial cell walls is enhanced by its insensitivity to specific chelating agents, allowing it to be utilised in combination with EDTA and lysozyme. However, the peptide interbridges of the cell walls of different species, formed by different combinations of component amino acids, with inherently different susceptibilities to cleavage, may be more or less resistant to Proteinase K lysis. While lysozyme and proteinase K are, probably, the enzymes most commonly used for the disruption of bacterial cells, additional bacterial cell-disrupting enzymes also have been reported with broad or narrow specificities. Other muramidases, mutanolysin and lysostaphin react, analogous to lysozyme, at the peptide linkages in the cell walls, although the species which are susceptible to these enzymes differ from those which are affected by lysozyme [2, 20, 26]. Subtilisins are extracellular proteases, produced by Bacillus spp., exhibiting a broad specificity in hydrolysing most peptide and ester bonds [24]. They are not inactivated MMEM-1.01/4 MMEM-1.01/5 MMEM-1.01/8 Figure 3. The recovery of DNA as a function of the amount of DNA in suspension. The recovery of DNA was observed to be dependent on the concentrations of the suspensions. The values indicated represent the means, calculated from the observed recoveries from suspension, after varying centrifugation times. The ranges of observed recoveries are indicated, with the lowest and highest recoveries, for each DNA concentration, corresponding to the shortest and longest centrifugation times (5-30 minutes). The graph was prepared from data taken from Zeugin and Hartley, 1985 [27]. Procedures The specific methods described here are simplified, rapid, protocols observed to be effective for isolating genomic DNA, from a wide range of bacteria, of a quality applicable for PCR. Protocol I-CTAB protocol for the extraction of bacterial genomic DNA This protocol is derived from the "miniprep" method described by Wilson [25]. Broth cultures (2-5 ml) grown to mid-log growth phase are harvested in 2.0 ml Eppendorf tubes by centrifugation in