Journal of Microbiological Methods 10 (1989) 59-64
Elsevier MIMET 00323
Simple enzymic method for isolation of DNA from diverse bacteria Shico Visuvanathan 1, Michael T. Moss:, John L. Stanford 1, John H e r m o n - T a y l o r 2 a n d J o h n J. M c F a d d e n 2 1Deoartment of Medical Microbiology, University College and Middlesex School of Medicine, London, UK; 2Department of Surgery, St. George's Hospital Medical School, London, UK
(Received 23 November 1988; revision received 14 April 1989; accepted 8 May 1989) Summary
An enzymic method for the extraction of high molecular weight DNA from a wide range of diverse bacteria, including mycobacteria, is described. The method is simple, rapid, requires few manipulations and is suitable for extracting quantitative yields of DNA from as few as --3 x 107 bacterial cells. The DNA produced is of good quality and is suitable for molecular biological manipulations. Key words: Bacterium; DNA extraction; Enzymic
Various protocols have been described for the extraction o f D N A from bacterial cells [ 1 - 4 ] . The critical step is lysis o f the bacterial cells, to release D N A whilst avoiding its degradation or shearing. E n z y m i c methods o f lysis [1, 2] produce high molecular weight D N A ; however, m a n y bacteria, such as mycobacteria, have cell walls that are resistant to enzymic lysis by the c o m m o n l y described methods. Physical and chemical methods o f lysis, such as grinding in liquid N, grinding with glass beads, passage t h r o u g h a H u g h e s press  or other cell disrupters or treatment with agents such as sodium triisopropylnaphthalene sulphonate and sodium 4-amino salicylate  are often employed but the D N A produced is often o f low molecular weight due to physical shearing or chemical degradation during extraction, and therefore unsuitable for m a n y molecular biological protocols. Additionally, large volumes
Correspondence to: M. T. Moss, St George's Hospital Medical School, Cranmer Terrace, London SW17ORE, UK
0167-7012/89/$ 3.50 © 1989 Elsevier Science Publishers BN. (Biomedical Division)
60 are often necessary, resulting in low yields when only small numbers of bacteria are available. We have developed a simple method to extract high molecular weight D N A from mycobacteria , modified from that described by Patel et al. . We now show that this method can be successfully applied to a wide range of diverse bacterial species, producing good yields of high molecular weight DNA, suitable for restriction endonuclease digestion and cloning. The method is simple, requires few manipulations, is rapid and can be applied to very small numbers of bacterial cells. We suggest its use as a general method of D N A extraction from bacteria for which specific protocols have not been developed and/or only very small quantities of bacterial cells are available. Methods
Bacterial strains and culture conditions A total of 68 bacterial strains are listed in Table 1. Mycobacterialspecies. The strains examined were obtained from J. L. Stanford. The original respective sources are listed in Table 1. All mycobacteria were subcultured onto Lowenstein-Jensen medium and incubated at 35 °C for the appropriate incubation periods and then harvested as described below. M. avium was grown in Middlebrook 7H9 broth with OADC enrichment. Other bacterial species. All NCTC, NCIB, IAM, ATCC and VPI strains were obtained freeze-dried. Clinical isolates were subcultured onto Columbia blood agar. Corynebacterium, Propionibacteriurn and Rhodococcus species were first cultured on Corynebacteriurn agar (Tryptone soya broth 30 g, yeast extract 10 g, agar no. 1 12 g and Tween 80 5 ml, .1 -~) at 37°C. All other isolates were grown on Columbia blood agar at 37 °C. DNA extraction Cells ( ~ 200 mg wet weight) were harvested from liquid culture by centrifugation at 5000 x g for 5 min and resuspended in 1 ml T E N buffer (50 m M T r i s - HC1, p H 8.0; 150 m M NaC1; 100 m M EDTA) in E p p e n d o r f tubes; or scraped off solid media, into E p p e n d o r f tubes containing T E N buffer (1 ml). Cells were recovered by centrifugation for 5 min (5000xg) in a benchtop microcentrifuge and resuspended in 0.4 ml TEN. To minimise biological hazard, the bacteria were incubated at 70 °C for 1 h. Fifty ~1 subtilisin (100 m g . m l -l, Sigma type VIII) was then added and the mixture was incubated at 37 °C for 18 h after which 100 #1 lysozyme (200 m g . m l 1, Sigma) was added and the mixture was incubated at 50 °C for 5 h. Lysis was completed by the addition of 50 #I 20% (w/v) SDS and 50 ~1 pronase (30 m g . m l -~, Calbiochem). The mixture was further incubated for 20 h, with the addition of a further 50 ~1 pronase after 12-15 h. The sample was spun for 5 min in a benchtop microcentrifuge to pellet the cell debris. The supernatant was recovered with a fine-tipped pipette with care being taken not to disturb the pellet.
61 An equal volume phenol : chloroform : isoamyl alcohol (50 : 48 : 2; v/v) saturated with TEN was added and the mixture was shaken by hand for 10 min. The phases were separated by centrifugation and the aqueous phase, containing the DNA, was carefully recovered. DNA was precipitated with 0.2 vol. 10 M ammonium acetate and 2 vols. 100% ethanol. After thorough mixing (by hand), the DNA could often be spooled. The spooled DNA was washed in 70% ethanol, dried briefly and resuspended (by gentle pipetting) in 100 t A T E (10 mM Tris-HCI, pH 8.0; 1 mM EDTA) containing 10 # g . m l - I boiled ribonuclease A (Sigma). Alternatively, the mixture was stored overnight at - 2 0 °C, the DNA recovered by centrifugation (10 min), washed in 70% ethanol and then dried and resuspended in TE as before. The DNA was stored at 4 °C for at least 24 h before use to ensure it had fully dissolved. DNA yield was determined spectrophotometrically [A260= 1= 50 ~g. ml-IDNA]. When extracting DNA from very small quantities of bacteria, the volumes and amounts were all reduced to one-fifth of the above and 5 t~l carrier glycogen (5 t~g'ml 1, Boehringer) was added to the resuspended cells before the addition of enzymes. DNA manipulations The approximate molecular weight of intact DNA was determined by electrophoresis of 200 ng DNA aliquots, in 0.8% agarose gels in TAE-running buffer (10 mM Tris-HC1; 5 mM sodium acetate; 0.5 mM EDTA, pH 7.8) containing ethidium bromide (0.5 t~g.ml-l), with Hind III-digested X phage DNA as a size marker. Restriction endonuclease digestions of DNA (0.2- 5.0/~g) were performed in 20-#1 vols., in accordance with the manufacturer's recommendations, but in buffers that additionally contained spermidine hydrochloride (4 ~g. ml-1). If the volume of test DNA exceeded 5 tA, then the digestion was performed in 100-200 #1 and the DNA was then precipitated (0.1 vol. 2 M NaC1, 2 vols. 100% ethanol), pelleted by centrifugation, dried briefly and resuspended in 20 ~1TE. Digested DNA was electrophoresed through 1% agarose gels as above. Results
DNA of high molecular weight was extracted from all the bacteria examined (Table 1), with variable yields, and DNA samples digested with most restriction endonucleases (Fig. 1). When complete digestion was not obtained with one restriction endonuclease, another invariably produced complete digestion, indicating incomplete digestion may have been due to methylation. Discussion
This method is generally applicable to a wide range of bacterial species. All aerobic and anaerobic gram-positive and gram-negative bacteria and mycobacteria investigated produced high yields of good quality DNA suitable for molecular biological manipulations. The protocol requires few manipulations and is rapid. The method is also highly reproducible.
62 TABLE 1 DNA YIELDS FROM B A C T E R I A L STRAINS Organism
Aeromonas hydrophila A lcaligenes odorans Arthrobacter globiformis Bacillus subtilis BacteroMes asaccharolyticus Bacteroides fragilis Bacteroides vulgatus Brevibacterium lipolyticum Cellulomonas biazotea Cellulomonas fimi Campylobacter jejuni Campylobacter pylori CIostridium difficile CIostridium perfringens CIostridium septicum Corynebacterium JK Corynebacterium minutissimum Enterobacter cloacae Escherichia coli Eubacterium aerofaciens Eubacterium lentum Fusobacterium varium Gardinerella vaginalis Klebsiella pneumoniae Lactobacillus casei var.rhamnosus Listeria monocytogenes Mycobacterium asiaticum Mycobacterium avium Mycobacterium bovis BCG Mycobacterium chitae Mycobacterium diernhoferi Mycobacteriurn duvalii Mycobacterium flavescens Mycobacterium fortuitum Mycobacteriurn gastri Mycobacterium gilvum Mycobacterium gordonae Mycobacteriurn intracellulare Mycobacterium kansas# Mycobacteriurn malmoense Mycobacterium marinum Mycobacterium neoaurum Mycobacterium nonchromogenicum Mycobacterium paratuberculosis Mycobacterium rhodesiae Mycobacterium smegmatis Mycobacterium therrnoresistibile Mycobacterium xenopi Mycoplasma species
Porton Down 13109 NCTC 10388 NCIB 8907 C1 NCTC 9337 NCTC 8343 NCTC 10583 IAM 1413 NCIB 8077 NCTC 7547 C1 CI CI C1 CI NCTC 11915 NCTC 10284 CI NCTC 10418 NCTC 11838 NCTC 11813 NCTC 10560 C1 Ct NCIB 8010 CI Institute Pasteur NCTC 8559 Glaxo strain ATCC 19630 El NCTC 358 NCTC 10270 NCTC 8573 NSCI CI NCTC 9822 ATCC 13950 CI NCTC 11299 CI NCTC 10439 El VI ATCC 27024 NCTC 333 ATCC 19528 NSCI CI
Yield* (~g D N A . g 625 294 333 115 192 225 582 149 275 150 267 156 108 134 110 167 370 200 215 145 250 111 93 66 182 128 53 76 125 54 64 59 84 97 63 179 54 73 104 208 97 130 106 78 128 78 116 99 76
I wet wt cells)
63 T A B L E 1 Continued Organism
Yield* (~g D N A . g - 1 wet wt cells)
Nocardia asteroides Nocardia brasiliensis Peptococcus asaccharolyticus Peptostreptococcus magnus Propionibacterium acnes Propionibacterium avidum Proteus mirabilis Pseudomonas aeruginosa Rhodococcus corallinus Rhodococcus species Rothia dentocariosa Salmonella enteriticlis Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus milleri Streptococcus pneumoniae Wolinella recta Yersinia enterocolitica
A T C C 3318 IMRV 800 N C T C 11461 CI VPI 0162 VPI 0589 C1 NCTC 10662 N C T C 10668 NCIB 9784 N C T C 10207 C1 N C T C 6571 CI CI CI C1 N C T C 11489 CI
124 111 185 159 58 50 189 182 44 29 233 263 84 63 84 35 55 149 383
* Corrected to nearest/~g. C1, clinical isolate; NSCI, nonsignificant clinical isolate; El, environmental isolate; VI, veterinary isolate.
Fig. 1. D N A samples electrophoresed through a 1% agarose gel and stained with ethidium bromide. Lanes 1- 5,200-ng aliquots of undigested D N A samples. Lanes 7 - 11, 5-#g aliquots o f PvuI 1-digested D N A samples, showing complete digestion. Bacteriophage k D N A digested with HindlII was used as a size marker (Lane 6). Lanes: (1, 7) Staphylococcus epidermidis, (2, 8) Fusobacterium varium, (3, 9) Clostridium perfringens, (4, 10)Mycobacterium kansasii, (5, 11)Nocardia asteroides.
64 DNA was also extracted by this method from Candida albicans (a yeast) and similar results were obtained. Further work with mycobacteria has shown that the enzyme incubation steps (18 h subtilisin, 5 h lysozyme, 20 h pronase) can be considerably reduced (i.e., 2 h subtilisin, 4 h lysozyme, 12 h pronase) without loss of yield (data not shown) and it is likely that this would also be found for most other bacteria. Since this simple method works for this diverse range of bacteria, including some thought resistant to lysis (i.e., mycobacteria), it should prove useful in extracting DNA from bacterial species for which specific extraction methods have not been described. The method is also suitable for extracting DNA from very small quantities of bacteria; we have used this method to extract 100 ng DNA from = 3 × 107 M. avium cells (representing a theoretical yield of 65 %). The method may also be a simple alternative where previously published methods are complicated, produce low yields or result in DNA of poor quality.
Acknowledgements We thank Miss Joe Thompson, Dr Liz Hull and Mr S Hampson for help in the development of the method used. Also Mr H. Hammill and Mr G. McIntyre for kind co-operation and help in acquiring bacterial strains and Dr J. Holton for helpful discussions and critical reading of the manuscript.
References 1 Marmur, J. (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3, 208-218. 2 Patel, R., Kvach, J. T. and Mounts, P. (1986) Isolation and restriction endonuclease analysis of mycobacterial DNA. J. Gen. Microbiol. 132, 541-551. 3 Hughes, D.E. (1951) A press for disrupting bacteria and other micro-organisms. Br. J. Exp. Pathol. 32, 97-109. 4 Hopwood, D. A., Bibb, M. J., Chater, K. F., Bruton, C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. and Schrempf, H. (1985) In: Genetic Manipulation of Streptomyces: a Laboratory Manual, John Innes Foundation, Norwich. 5 McFadden, J.J., Butcher, RD., Chiodini, R. and Hermon-Taylor, J. (1987) Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species. J. Clin. Microbiol. 25, 796-801.