Jan 16, 1989 - terial genomic DNA with guanidium thiocyanate. Letters in ... aerophilic atmosphere (Owen & Dawson 1986) and the .... Chichester: John Wiley.
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Letters in Applied Microbiology 1989, 8, 151-156
Rapid extraction of bacterial genomic DNA with guanidium thiocyanate D . G . P I T C H E RN.A. , S A U N D E R S&* R . J . O W E NNational Collection of T y p e Cultures and *Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, London N W 9 5HT, U K Received 16 January 1989 and accepted 18 January 1989 PITCHER. D . G . . SAUNDERS. N . A . SLOWEN. R . J . 1989. Rauid extraction of bacterial genomic DNA with guanidium thiocyanate. Letters in Agplied Microbiology 8, 151-156 A method is described for the rapid isolation and purification of bacterial genomic DNA. A total of 215 bacterial strains representing species of Campylobacter, Corynebacterium, Escherichia, Legionella, Neisseria, Staphylococcus and Streptococcus, were lysed with guanidium thiocyanate. DNA was prepared using just three other reagents and one high-speed centrifugation step. The method, which was applicable to both Gram-positive and Gram-negative bacteria, eliminated endogenous nuclease activity and avoided the need for phenol, RNase and protease treatments. The DNA was of high purity, high molecular mass and double-stranded.
Various methods have been described for the rapid isolation of chromosomal DNA from prokaryotic cells (Beji et al. 1986; Weeks et al. 1986; Dawson et al. 1987; Lewington et al. 1987; Owen & Borman 1987; Yacoob & Zealey 1988). In this report we describe a simple method of extracting DNA using guanidium thiocyanate, a strong protein denaturant, which has been used to lyse a variety of both prokaryotic and eukaryotic cells for nucleic acid extraction, particularly when high nuclease activity was a problem (Chirgwin et al. 1979). The method also involves selective precipitation of DNA with 2-propanol in 2.5 mol/l ammonium acetate (Owen & Borman 1987). The method is applied to both Gram-positive and Gramnegative bacteria.
lobacter jejuni (15), Pseudomonas aeruginosa (1 5), Campylobacter pylori (lo), Neisseria meningitidis (9), Escherichia coli (2), and Streptococcusfaecalis (1). The strains of Escherichia, Neisseria, Pseudomonas, Staphylococcus and Streptococcus were cultured aerobically at 37°C in 20 ml Tryptone Soya Broth (Oxoid) supplemented with 0.4% w/v yeast extract. Tween 80 (0.2% v/v) was added to the medium for the culture of C . jeikeium. Campylobacter strains were grown on 5% v/v horse blood agar at 37°C in a microaerophilic atmosphere (Owen & Dawson 1986) and the Legionella strains were grown on BCYE agar at 37°C for 48 h (Dournon 1988). The strains were obtained from the National Collection of Type Cultures and the Division of Microbiological Reagents and Quality Control. D N A EXTRACTION
Materials and Methods BACTERIA A N D G R O W T H CONDITIONS
Strains of the following bacterial species (numbers tested in parentheses) were used: Legionella pneumophila (loo), Corynebacterium jeikeium (38), Staphylococcus aureus (25), Campy-
Broth cultures were harvested at the end of the exponential growth phase by centrifugation at lo00 g for 15 min. A small (rice grain-sized) cell pellet was obtained. The Campylobacter and Legionella cultures were harvested with a plastic spatula from the surface of plate media to give a similar sized pellet. The cells of Gram-positive
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species were resuspended in 100 p1 of fresh lysozyme (50 mg/ml) in TE buffer (10 mmol Tris-HC1; 1 mmol/l EDTA, pH 8). Cells of Staph. aureus were suspended in 100 pl lysostaphin (50 mg/ml) in 0.1 mol/l sodium phosphate at pH 7. The suspensions were incubated at 37°C for 30 min. The Gram-negative species were resuspended in 100 p1 of TE buffer without enzymic treatment. Cells were lysed with 0.5 ml 5 mol/l guanidium thiocyanate (Sigma), 100 mmol/l EDTA and 0.5% v/v sarkosyl (GES reagent), which was prepared as follows. Guanidium thiocyanate (60 g), 0.5 mol/l EDTA at pH 8 (20 ml) and deionized water (20 ml) were heated at 65°C with mixing until dissolved. After cooling, 5 ml of 10% v/v sarkosyl were added, the solution was made up to 100 ml with deionized water, filtered through a 0.45 pm Nalgene filter (BDH Ltd) and stored at room temperature. Cell suspensions were vortexed briefly and checked for lysis (5-10 min). The lysates were cooled on ice, 0.25 ml cold 7.5 mol/l ammonium acetate added with mixing, held on ice for a further 10 min and then 0.5 ml chloroform and 2-pentanol (24: 1) mixture added. The phases were mixed thoroughly, transferred with a widebore pasteur pipette to a 1.5 ml Eppendorf tube and centrifuged (25000 g) for 10 min. Supernatant ffuids were transferred to Eppendorf tubes and 0.54 volumes of cold 2-propanol added. The tubes were inverted for 1 min to mix the solutions and the fibrous DNA precipitate was deposited by centrifugation at 6500 g for 20 s. Pellets of DNA were washed five times in 70% ethanol and dried under vacuum. DNA CHECKS
DNA samples were redissolved overnight at 4°C in a small volume (usually 100 pl) of sterile, deionized water. The 280: 260 nm absorbance ratio, hyperchromicity and melting temperature were determined in 0.1 x SSC (SSC = 150 mmol/l NaCI, 15 mmol/l trisodium citrate) (Owen & Pitcher 1985). The integrity of the DNA (10 pl samples) was checked by horizontal gel electrophoresis in 0.5% agarose in TBE buffer (89 mmol/l Tris, 89 mmolb boric acid, 2 mmol/l EDTA, pH 8.3) containing ethidium bromide (0.5 mg/ml). The DNA was digested with ClaI, EcoRI, PuuII, HindIII, SalI or SmaI (BRL-Gibco; BCL) for 4 h and the resulting
fragments were separated by horizontal electrophoresis in 0.8% agarose gels in TBE at 30 V for 20 h. Preparation of ribosomal RNA gene probes, Southern blot hybridization and detection of hybrid bands were performed as described previously (Pitcher et al. 1987; Owen et al. 1988). Results and Discussion DNA was extracted from 215 strains representing nine different bacterial species. All the Gram-negative bacteria lysed within 5 min of mixing with the GES reagent but lysis of Grampositive bacteria was slower and yields of DNA were low. This problem was resolved by introducing an additional step in which the cell pellets were first treated with either lysozyme or, in the case of Staph. aureus, lysostaphin. The lysis of Gram-positive cells was then completed within 5 min. Recent studies have shown that the method also enabled DNA to be obtained readily from Listeria monocytogenes, which proved problematic when other extraction methods were used (unpublished results). An important advantage of using guanidium thiocyanate is that it inactivates endogenous nucleases. The 2.5 mol/l ammonium acetate salts-out proteins and stabilizes nucleic acids, ensuring a firm band of insoluble protein and cell debris was formed at the aqueous liquid/ chloroform interphase and allowing the removal of the relatively viscous DNA solution (the upper phase) without disturbing the interphase. Deproteinization with GES was highly effective as additional chloroform treatments did not precipitate any further protein. Deproteinated supernatant fluids were clear and sometimes straw-coloured, and when 0.54 volumes of 2propanol were added with gentle mixing, fibrous strands of DNA became visible. Addition of exactly the correct amount of 2-propanol ensured that only high molecular mass DNA was precipitated (Raeder & Broder 1985). Electrophoresis in agarose gels showed that the DNA obtained was of a high molecular mass and relatively undegraded (Fig. 1). In the case of the Staph. aureus DNA sample, traces of plasmid DNA were detected when the gel was heavily loaded. However, these plasmids were removed by redissolving the DNA in 2.5 molb ammonium acetate and re-precipitating with 2propanol and 70% ethanol. In some cases traces
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Fig. 1. Native DNA prepared with GES reagent after electrophoresis in 0.5% agarose in TBE buffer for 3 h at 5 V/cm. Sample loaded: a, 1-2 jig; b, 1&20 pg. Lane: 1, Staphylococcus aureus NCTC 10442; 2, Pseudomonas aeruginosa NCTC 10332; 3, Corynebacteriumjeikeiurn NCTC 11913; 4, Escherichia coli NCTC 9001.
of ribosomal RNA were also detected. As the DNA fibres obtained after 2-propanol precipitation readily adhered to the walls of the Eppendorf tubes, washing steps were carried out without high-speed centrifugation. This avoided impacting the DNA and making it difficult to redissolve.
The physical characteristics of DNA from selected strains showed that the samples were essentially free from contaminating proteins, RNA and other molecules absorbing at 260 nm (Table 1). Thermal denaturation resulted in hyperchromic shifts of about 30%, which were characteristic of pure, double-stranded DNA.
Table 1. The physical characteristics of representative DNA samples purified by the GES reagent method A,,, Species
Strain*
Escherichia coli Pseudomonas aeruginosa Corynebacteriurnjeikeium StaDhvlococcus aureus
NCTC 9001T NCTC 10332T NCTC 11913' NCTC 10442
A,,,
",/ nm
1.79t 1.96 1.82 1.97
G + C content (mol Yo) Hyperchromic shift (Yo) 29 31 31 25
Estimated
Literature
51 66
51 (I)$ 66 (2) 60 (3) 32-36 14)
60 33
* T, Type strain.
t A ratio of about 1.8 is exmcted for Dure DNA (Maniatis et al. 19821.
3 Values for strains or species range were from the following publications: 1, Owen et al. (1987); 2, Tamaoka & Kamagata (1984); 3, Jackman et al. (1987); 4, Schleifer (1986).
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Fig. 2. Electrophoretic patterns of restriction enzyme digests of DNA prepared with GES reagent. Pseudomonas aeruginosa NCTC 10332 with Hind11 (lane 1)and PuuII (lane 2). Cumpylobacter pylori NCTC 11639 with HindIII (lane 3). Neisseria meningitidis NCTC 10026 with ClaI (lane 4) and PuuII (lane 5). Streptococcus faecalis NCTC 775 with Hind111 (lane 6) and PuuII (lane 7). Corynebacterium jeikeium NCTC 11913 with HindIII (lane 8) and PuuII (lane 9). Bacteriophage lambda (Gibco-BRL) with HindIII (lane 10).
Estimated base compositions (mol % G + C) were within 1% of the values found previously for those species using traditional DNA isolation methods (Marmur 1961; Owen & Pitcher 1985). When the DNA was digested with restriction enzymes, characteristic band patterns were obtained (Fig. 2) showing that the DNA samples were double-stranded, and free of residual guanidium thiocyanate and other enzyme-inhibiting molecules. Southern hybridization with specific gene probes produced discrete bands against clear backgrounds (Fig. 3). We conclude that the guanidium thiocyanate extraction method is rapid and convenient for
purifying DNA in high yields from small quantities of Gram-positive and Gram-negative bacterial cells.
References BEJI,A., IZARD, D., GAVINI,F., LECLERC, H., LESEINEM. & KREMBEL, J. 1986 A rapid DELSTANCHE, chemical procedure for isolation and purification of chromosomal DNA from Gram-negative bacilli. Analytical Biochemistry 162, 18-23. CHIRGWIN, J.M., PRZYBLA, A.E., MACDONALD, R.J. & RUTTER,W.J. 1979 Isolation of biologically active ribonucleic acid from sources rich in ribonuclease. Biochemistry 18,5294-5299.
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Fig. 3. Southern blot hybridization patterns of restriction enzyme digests of DNA prepared with GES reagent from a, Corynebacterium jeikeium NCTC 11913 with Hindi11 (lane I), EcoRI (lane 2) and PuuII (lane 3); and b, Pseudomonas aeruginosa NCTC 10332 with Hind111 (lane l), EcoRI (lane 2) and Smal (lane 3). The probes used were biotinylated cDNA transcribed from total rRNA from each strain.
DAWSON,C., OWEN,R.J. & BECK,A. 1987 A method for rapid extraction of Prouidencia chromosomal DNA and restriction enzyme digestion in agarose pellets. Letters in Applied Microbiology 5 , 4 1 4 . DOURNON, E. 1988 Isolation of Legionella from clinical specimens. In A Laboratory Manual for Legionella ed. Harrison, T.G. & Taylor, A.G. pp. 13-30. Chichester: John Wiley. D.G., PELCZYNSKA, S. & JACKMAN, P.J.H., PITCHER, BORMAN, P. 1987 Classification of corynebacteria associated with endocarditis (group JK) as Corynebacterium jeikeium sp. nov. Systematic and Applied Microbiology 9,83-90. LEWNGTON,J., GREENAWAY, S.D. & SPILLANE, B.J. 1987 Rapid small scale preparation of bacterial genomic DNA suitable for cloning and hybridization analysis. Letters in Applied Microbiology 5, 51-53. MANIATIS,T., FRITSCH E.F. & SAMBROOK, J. 1982 Molecular Cloning: A hboratory Manual Cold Spring Harbor: Cold Spring Harbor Laboratory. MAMUR,J. 1961 A procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of Molecular Biology 3,208-218.
OWEN,R.J. & PITCHER, D. 1985 Chemical methods for estimating DNA base compositions and levels of DNA-DNA hybridization. In Chemical Methods in Bacterial Systematics ed. Goodfellow, M. & Minnikin, D.E. pp. 67-93. London: Academic Press. OWEN,R.J. & DAWSON,C. 1986 DNA base composition and base sequence relatedness of atypical Campylobacter jejuni strains from clinical material. FEMS Microbiology Letters 35,283-287. OWEN,R.J. & BORMAN,P. 1987 A rapid biochemical method for purifying high molecular weight bacterial chromosomal DNA for restriction enzyme analysis. Nucleic Acids Research 15, 3631. OWEN, R.J., AHMED, A.U. & DAWSON,C.A. 1987 Guanine-plus-cytosine contents and type strains of the genus Providencia. International Journal of Systematic Bacteriology 37,449-450. OWEN, R.J., BECK,A., DYAL,P.A. & DAWSON,C. 1988 Detection of genomic variation within Prooidencia stuartii clinical isolates by analysis of DNA restriction fragment-length polymorphisms containing ribosomal RNA cistrons. Journal of Clinical Microbiology 26,2161-2166. PITCHER,D.G., OWEN,R.J., DYAL,P. & BECK,A. 1987 Synthesis of a biotinylated DNA probe to detect
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ribosomal RNA cistrons in Prooidencia stuartii. FEMS Microbiology Letters 48,283-287. RAEDER,U. & BRODER,P. 1985 Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17-20. SCHLEIFER,K.H. 1986 Family 1. Micrococcaceae Prevot 1961,31AL.In Bergey’s Manual ofSysternatic Bacteriology Vol. 2 ed. Sneath, P.H.A., Mair, N.S., Sharpe, M.E. & Holt, J.G. pp. 1003. Baltimore: Williams & Wilkins. TAMAOKA, J. & KOMACATA, K. 1984 Determination of DNA base composition by reversed-phase high per-
formance liquid chromatography. FEMS Microbiology Letters 25, 125-128. WEEKS,D.P., BEERMAN, N. & GRIFFITH, O.M. 1986 A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: applications to isolations from mammalian, insect, higher plant, algal, yeast and bacterial sources. Analytical Biochemistry 152, 376385. YACOOB,R.K. & ZEALEY,G.R. 1988 A one-step procedure for the purification of high molecular weight bacterial chromosomal DNA. Nucleic Acids Research 16, 1639.
