Bacterial Cell Lysis and Release of Intact Chromosomal DNA

A pure culture of a bacterial isolate of known identity is incubated overnight in a nutrient broth, such as trypticase soy broth (BD Biosciences, Sparks, MD, USA) in order to achieve 109cells/mL, which is the concentration needed to obtain a visible band pattern. Standard DNA extraction procedures are inappropriate for the analysis of large chromosomal DNA molecules because of DNA shearing caused by the application of mechanical force in the protocol. In order to prevent DNA damage, it is important to mix intact bacterial cells with warmed, liquid-phase agarose, and this mixture can then be pipetted into plastic molds to form 10 x 5 x 1.5 mm agarose plugs. The whole cells embedded in plugs are lysed and deproteinized by detergents and enzymes (e.g., lysostaphin, lysozyme, proteinase K, mutanolysin or lyticase) in situ (Table 9.1). Following cell lysis, the plugs are washed 4-5 times with wash buffer containing 20 mM Tris and 50 mM EDTA (pH 8.0) to remove cell debris and proteinase. The agarose gel matrix keeps chromosomal DNA intact while removing the rest of cellular components from the plug. This step, including the post-lysis washing, usually takes 2 days. In recent years, several investigators have reported improvements to the traditional DNA preparation process. The most notable time-saving approaches have included (i) directly using bacterial colonies grown on plates of clinical specimens, (ii) using a combination of lytic enzymes, (iii) adding lytic enzymes to the bacterial suspensions before preparing the agarose plugs, (iv) shortening the cell lysis time by reducing the size of agarose plug, and (v) expediting the wash steps by using a large volume (10 mL) of preheated (50°C) water and TE buffer (Gautom, 1997; Turabelidze et al., 2000; Lopez-Canovas et al., 2003). Of particular interest, a DNA purification system was designed to automate all steps involved in the preparation of DNA plugs for PFGE (Fiett et al., 2004).

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