Nucleic Acids Research, Vol. 19, No. 8 1955

Q-D/ 1991 Oxford University Press

A simple method for the isolation of chromosomal DNA from Gram positive or acid-fast bacteria C.Bollet*, M.J.Gevaudan, X.de Lamballerie, C.Zandotti and P.de Micco Laboratoire de Microbiologie et d'Hygiene Hospitalieres, HOpital Salvator, Marseille F13009, France Submitted January 2, 1991 Current methods for isolation of DNA from Gram positive or acid-fast bacteria lack efficiency and are time-consuming: chemical disruption of the cells is tedious due to the cell-wall thickness. Mechanical lysis procedures (French press, Mini-Beadbeater, Ribi pressure cell) require specific equipment and can damage the DNA (1). Culture with glycin, ampicillin, or cycloserin to obtain protoplasts is fastidious and usually requires numerous successive experiments to adapt the technique to the different bacterial strains (2). Successive enzymatic lysis Oysozyme, lysostaphin, pronase, proteinase K) is time-consuming. Moreover, these enzymes lack efficacy on most Gram positive bacteria: lysozyme is inactive on staphylococci and most Bacillus strains. Lysostaphin is active only on staphylococci. Proteinase K is very efficient on Gram negative bacteria (3, 4), but lacks efficiency on Gram positive bacteria. In addition, proteinase K is very expensive. Achromopeptidase is active on most Gram positive bacteria (Corynebacterium and Mycobacterium excepted) but usually shears DNA (5). Finally, the most efficient techniques are thermal shock (-196°C/+100°C) and SDS (3% final concentration), which make it possible to obtain 5 to 50 tg of DNA from a pellet of 200 mg of wet weight of bacteria. However, Mycobacterium and Corynebacterium are resistant. By using an ordinary micro-wave oven, we were able to develop a one-hour procedure which routinely yields 20 to 200 isg of high molecular weight DNA from a pellet of 200 mg of wet weight of bacteria. Strains were grown on Middlebrook medium (mycobacteria), Columbia + 5% sheep blood (streptococci, anaerobes) or chocolate agar slant (other bacteria). Bacteria were collected and pelleted in a microfuge, washed with 1 ml TE (10 mM Tris pH 8, 10 mM EDTA) and resuspended in 100 IL TE. 50 $1 of 10% SDS were added and the solution was incubated 30 minutes at 65°C. The lysate was centrifuged and the supematants were removed. The microtubes were then placed in a micro-wave oven and heated for 2 x 1 minute (900 W) or 3 x 1 minute (750 W). The pellets were dissolved in 200 yd of TE and shaken with an equal volume of chloroformisoamylic alcohol-phenol (24:1:25) for 15 minutes. The aqueous phase was recovered by centrifugation and precipitated in ehanol (centrifugation: 20 minutes). Plasmid DNA is not spoiled and can be extracted by the alkali method: chromosomal DNA is denatured during 30 minutes at

*

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70°C in 50 mM Tris, 80 mM NaOH, pH 12.6. After neutralization (3 M sodium acetate, pH 4.8), the plasmid DNA can be recovered in the supernatant. Numerous plasmids from Corynebacterium, Clavibacter, Staphylococcus and Mycobacterium were recovered using this procedure. The only variable parameter is the duration of heating. It depends on the oven and the wet weight of the bacteria. Once this duration has been established, the procedure could be applied to all bacterial genera. This method is of wide interest in Corynebacterium, Actinomyces and Nocardia: using the microwave considerably increases the amount of extracted DNA (Table 1). The extracted DNA usually has a molecular weight of 20 kb and is suitable for restriction, ligation or PCR. Table 1. Yield of DNA obtained from different strains using simple 3% SDS lysis or with supplementary micro-wave heating. Bacterial strains

amount of DNA (ug/200 mg of wW) 3% SDS 3% SDS + MWO

Mycobacterium tuberculosis Actinomyces israelii Nocardia asteroides Streptococcus pyogenes Rhodococcus equi Bacillus gordonae Corynebacterium xerosis Clostridium perfringens

0 10 10 50 5 5 0 20

50 200 100 200 50 50 20 150

REFERENCES 1. Hurley,S.S., Splitter,G.A. and Welch,R.A. (1987) J. Clin. Microbiol. 25, 2227-2229. 2. Heath,L.S., Sloan,G.L. and Heath,H.E. (1986) AppL. Environ. Microbiol. 51, 1138-1140. 3. Grimberg,J., Maguire,S. and Belluscio,L. (1989) Nucl. Acids Res. 17, 8893. 4. Grimberg,J., Nawoschik,S., Belluscio,L., McKee,R., Turck,A. and Eisenberg,A. (1989) Nucd. Acids Res. 17, 8390. 5. Barsotti,O., Renaud,F., Freney,J., Benay,G., Decoret, D. and Dumont,J. (1989) Ann. Inst. Pasteur/Microbiol. 138, 529-536.

A simple method for the isolation of chromosomal DNA from gram positive or acid-fast bacteria.

Nucleic Acids Research, Vol. 19, No. 8 1955 Q-D/ 1991 Oxford University Press A simple method for the isolation of chromosomal DNA from Gram positiv...
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