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Letters in Applied Microbiology 1989, 8, 151-156 CRH/OOI

Rapid extraction of bacterial genomic DNA with guanidium


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

D . G . . SAUNDERS. R . J . 1989. Rauid extraction of bac-
terial genomic DNA with guanidium thiocyanate. Letters in Agplied Microbiology 8,
A method is described for the rapid isolation and purification of bacterial genomic
DNA. A total of 215 bacterial strains representing species of Campylobacter, Cory-
nebacterium, 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 nucle-
ase 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 lobacter jejuni (15), Pseudomonas aeruginosa (1 5),
rapid isolation of chromosomal DNA from pro- Campylobacter pylori (lo), Neisseria meningitidis
karyotic cells (Beji et al. 1986; Weeks et al. (9), Escherichia coli (2), and Streptococcusfaecalis
1986; Dawson et al. 1987; Lewington et al. (1). The strains of Escherichia, Neisseria,
1987; Owen & Borman 1987; Yacoob & Zealey Pseudomonas, Staphylococcus and Streptococcus
1988). In this report we describe a simple were cultured aerobically at 37°C in 20 ml
method of extracting DNA using guanidium Tryptone Soya Broth (Oxoid) supplemented
thiocyanate, a strong protein denaturant, which with 0.4% w/v yeast extract. Tween 80 (0.2%
has been used to lyse a variety of both prokary- v/v) was added to the medium for the culture of
otic and eukaryotic cells for nucleic acid extrac- C . jeikeium. Campylobacter strains were grown
tion, particularly when high nuclease activity on 5% v/v horse blood agar at 37°C in a micro-
was a problem (Chirgwin et al. 1979). The aerophilic atmosphere (Owen & Dawson 1986)
method also involves selective precipitation of and the Legionella strains were grown on BCYE
DNA with 2-propanol in 2.5 mol/l ammonium agar at 37°C for 48 h (Dournon 1988). The
acetate (Owen & Borman 1987). The method is strains were obtained from the National Collec-
applied to both Gram-positive and Gram- tion of Type Cultures and the Division of
negative bacteria. Microbiological Reagents and Quality Control.

Materials and Methods
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
Strains of the following bacterial species pellet was obtained. The Campylobacter and
(numbers tested in parentheses) were used: Legionella cultures were harvested with a plastic
Legionella pneumophila (loo), Corynebacterium spatula from the surface of plate media to give a
jeikeium (38), Staphylococcus aureus (25), Campy- similar sized pellet. The cells of Gram-positive
152 D. G. Pitcher et al.
species were resuspended in 100 p1 of fresh fragments were separated by horizontal electro-
lysozyme (50 mg/ml) in TE buffer (10 mmol phoresis in 0.8% agarose gels in TBE at 30 V
Tris-HC1; 1 mmol/l EDTA, pH 8). Cells of for 20 h. Preparation of ribosomal RNA gene
Staph. aureus were suspended in 100 pl lyso- probes, Southern blot hybridization and detec-
staphin (50 mg/ml) in 0.1 mol/l sodium phos- tion of hybrid bands were performed as
phate at pH 7. The suspensions were incubated described previously (Pitcher et al. 1987; Owen
at 37°C for 30 min. The Gram-negative species et al. 1988).
were resuspended in 100 p1 of TE buffer without
enzymic treatment. Cells were lysed with 0.5 ml
Results and Discussion
5 mol/l guanidium thiocyanate (Sigma), 100
mmol/l EDTA and 0.5% v/v sarkosyl (GES DNA was extracted from 215 strains represent-
reagent), which was prepared as follows. Gua- ing nine different bacterial species. All the
nidium thiocyanate (60 g), 0.5 mol/l EDTA at Gram-negative bacteria lysed within 5 min of
pH 8 (20 ml) and deionized water (20 ml) were mixing with the GES reagent but lysis of Gram-
heated at 65°C with mixing until dissolved. positive bacteria was slower and yields of DNA
After cooling, 5 ml of 10% v/v sarkosyl were were low. This problem was resolved by intro-
added, the solution was made up to 100 ml with ducing an additional step in which the cell
deionized water, filtered through a 0.45 pm pellets were first treated with either lysozyme or,
Nalgene filter (BDH Ltd) and stored at room in the case of Staph. aureus, lysostaphin. The
temperature. lysis of Gram-positive cells was then completed
Cell suspensions were vortexed briefly and within 5 min. Recent studies have shown that
checked for lysis (5-10 min). The lysates were the method also enabled DNA to be obtained
cooled on ice, 0.25 ml cold 7.5 mol/l ammonium readily from Listeria monocytogenes, which
acetate added with mixing, held on ice for a proved problematic when other extraction
further 10 min and then 0.5 ml chloroform and methods were used (unpublished results).
2-pentanol (24: 1) mixture added. The phases An important advantage of using guanidium
were mixed thoroughly, transferred with a wide- thiocyanate is that it inactivates endogenous
bore pasteur pipette to a 1.5 ml Eppendorf tube nucleases. The 2.5 mol/l ammonium acetate
and centrifuged (25000 g) for 10 min. Super- salts-out proteins and stabilizes nucleic acids,
natant ffuids were transferred to Eppendorf ensuring a firm band of insoluble protein and
tubes and 0.54 volumes of cold 2-propanol cell debris was formed at the aqueous liquid/
added. The tubes were inverted for 1 min to mix chloroform interphase and allowing the removal
the solutions and the fibrous DNA precipitate of the relatively viscous DNA solution (the
was deposited by centrifugation at 6500 g for upper phase) without disturbing the interphase.
20 s. Pellets of DNA were washed five times in Deproteinization with GES was highly effective
70% ethanol and dried under vacuum. 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 2-
DNA samples were redissolved overnight at 4°C propanol were added with gentle mixing,
in a small volume (usually 100 pl) of sterile, fibrous strands of DNA became visible. Addi-
deionized water. The 280: 260 nm absorbance tion of exactly the correct amount of 2-propanol
ratio, hyperchromicity and melting temperature ensured that only high molecular mass DNA
were determined in 0.1 x SSC (SSC = 150 was precipitated (Raeder & Broder 1985).
mmol/l NaCI, 15 mmol/l trisodium citrate) Electrophoresis in agarose gels showed that
(Owen & Pitcher 1985). The integrity of the the DNA obtained was of a high molecular
DNA (10 pl samples) was checked by horizontal mass and relatively undegraded (Fig. 1). In the
gel electrophoresis in 0.5% agarose in TBE case of the Staph. aureus DNA sample, traces of
buffer (89 mmol/l Tris, 89 mmolb boric acid, 2 plasmid DNA were detected when the gel was
mmol/l EDTA, pH 8.3) containing ethidium heavily loaded. However, these plasmids were
bromide (0.5 mg/ml). The DNA was digested removed by redissolving the DNA in 2.5 molb
with ClaI, EcoRI, PuuII, HindIII, SalI or SmaI ammonium acetate and re-precipitating with 2-
(BRL-Gibco; BCL) for 4 h and the resulting propanol and 70% ethanol. In some cases traces
Rapid D N A extraction 153

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 The physical characteristics of DNA from
DNA fibres obtained after 2-propanol precipi- selected strains showed that the samples were
tation readily adhered to the walls of the essentially free from contaminating proteins,
Eppendorf tubes, washing steps were carried out RNA and other molecules absorbing at 260 nm
without high-speed centrifugation. This avoided (Table 1). Thermal denaturation resulted in
impacting the DNA and making it difficult to hyperchromic shifts of about 30%, which were
redissolve. characteristic of pure, double-stranded DNA.

Table 1. The physical characteristics of representative DNA samples purified by the GES reagent method
G + C content (mol Yo)
A,,, ",/ Hyperchromic
Species Strain* A,,, nm shift (Yo) Estimated Literature
Escherichia coli NCTC 9001T 1.79t 29 51 51 (I)$
Pseudomonas aeruginosa NCTC 10332T 1.96 31 66 66 (2)
Corynebacteriurnjeikeium NCTC 11913' 1.82 31 60 60 (3)
StaDhvlococcus aureus NCTC 10442 1.97 25 33 32-36 14)
* 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).
154 D. G. Pitcher et al.

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) purifying DNA in high yields from small quan-
were within 1% of the values found previously tities of Gram-positive and Gram-negative bac-
for those species using traditional DNA iso- terial cells.
lation methods (Marmur 1961; Owen & Pitcher
1985). When the DNA was digested with
restriction enzymes, characteristic band patterns References
were obtained (Fig. 2) showing that the DNA
samples were double-stranded, and free of DELSTANCHE, M. & KREMBEL, J. 1986 A rapid
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enzyme-inhibiting molecules. Southern hybrid- chromosomal DNA from Gram-negative bacilli.
ization with specific gene probes produced dis- Analytical Biochemistry 162, 18-23.
crete bands against clear backgrounds (Fig. 3). RUTTER,W.J. 1979 Isolation of biologically active
We conclude that the guanidium thiocyanate ribonucleic acid from sources rich in ribonuclease.
extraction method is rapid and convenient for Biochemistry 18,5294-5299.
Rapid D N A extraction 155

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.

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