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

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. N . A . SLOWEN.
D . G . . SAUNDERS. R . J . 1989. Rauid extraction of bac-
terial 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, 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
rapid isolation of chromosomal DNA from pro-
karyotic 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 prokary-
otic and eukaryotic cells for nucleic acid extrac-
tion, 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 Gram-
negative 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 micro-
aerophilic 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 Collec-
tion of Type Cultures and the Division of
Microbiological Reagents and Quality Control.

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-
D N A EXTRACTION
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
152 D. G. Pitcher et al.
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 lyso-
staphin (50 mg/ml) in 0.1 mol/l sodium phos-
phate 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. Gua-
nidium 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 wide-
bore pasteur pipette to a 1.5 ml Eppendorf tube
and centrifuged (25000 g) for 10 min. Super-
natant 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 electro-
phoresis in 0.8% agarose gels in TBE at 30 V
for 20 h. Preparation of ribosomal RNA gene
probes, Southern blot hybridization and detec-
tion 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 represent-
ing nine different bacterial species. All the
Gram-negative bacteria lysed within 5 min of
mixing with the GES reagent but lysis of Gram-
positive bacteria was slower and yields of DNA
were low. This problem was resolved by intro-
ducing 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 2-
propanol were added with gentle mixing,
fibrous strands of DNA became visible. Addi-
tion 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 2-
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
DNA fibres obtained after 2-propanol precipi-
tation 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
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)
were within 1% of the values found previously
for those species using traditional DNA iso-
lation 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 hybrid-
ization with specific gene probes produced dis-
crete 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 quan-
tities of Gram-positive and Gram-negative bac-
terial cells.
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 Rapid D N A extraction 155

Fig. 3. Southern blot hybridization patterns of restriction enzyme digests of DNA prepared with GES reagent
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