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Memoirs Kyushu Univ. Dep. Of Health Scis. Of Medical Sch.,2004,vol.3,33-38
Optimal Bacterial DNA Isolation Method
Using Bead-Beating Technique.
Shuji Fujimoto 1), Yoshiko Nakagami 1), Fumiko Kojima 1),2）
1) Department of Health Sciences, Graduate School of Medical Sciences, Kyusyu University
2) Graduate School of Medicine, Osaka University
ABSTRACT
Extraction of nucleic acids from Gram-positive
bacteria is normally hampered by a thick and
resistant cell wall. This paper presents
procedures based on mechanical cell breakage to
extract DNA from Staphylococcus aureus. The
proposed system for DNA extraction involves
bead-beating treatment and the GES method.
These two steps allow a consistent extraction
from the bacterium resistant to the GES method
alone. Yield and quality of DNA obtained with the
proposed method were higher than those
obtained with the GES method alone. This
protocol can be extended to clinical specimens.
Key words: bacterial DNA isolation, bead-beating
method and Staphylococcus aureus
INTRODUCTION
Genetic testing and diagnosis of infectious
diseases are playing an important role in the field
of clinical microbiology, today. This is a molecular
biology-based diagnostic method for detecting and
analyzing a base sequence of the nucleic acid
(DNA or RNA) that is specific to the target
microorganism, and the range of applications of
this method is broad and can be divided into two
categories as follows:
An application as a means of identifying
microorganisms directly in test samples without
culture, which has contributed to the speed up of
testing, improvement of detection sensitivity and
detection of microorganisms that may be difficult
or even dangerous to grow in culture. There have
been reports regarding the detection of bacterial
DNA in various types of clinical specimens, such
as blood and plasma１)２)，cerebrospinal fluid３)
４) and other test samples, by the use of this
method.
The other is an application in epidemiological
analysis ６ ) ７ ) to classify bacteria previously
isolated and cultured, detect and identify their
species and causative agents. In genetic testing of
bacterial pathogens, extraction of nucleic acids
from bacteria found in clinical specimens is the
first step. Using the nucleic acid obtained as
templates, target genes are amplified by the
Polymerase Chain Reaction (PCR) with various
primers and small amounts of nucleic acids are
amplified and detected by techniques, such as
hybridization using various probes.
The problem of these molecular biological
approaches is an extraction process of nucleic
acids from Gram-positive bacteria. To extract
nucleic acids from bacteria, bacterial cells are
destroyed to allow for release of nucleic acids
contained within the cytoplasm, and remaining
proteins should be removed with methods, such
as deproteinization. Gram-positive bacteria have
a thick cell wall consisting mainly of many layers
of peptidoglycan, which is not easily destroyed. In
general, extraction should be performed only after
bonds in the peptidoglycan have been cut using a
peptidoglycan nicking enzyme that is best suited
120124 Optimal Bacterial DNA using bead beating.doc
for
each
type
of
bacteria. For example,
Staphylococcus aureus bacteria should be treated
with lysostaphin for several hours at 37°C before
extraction is performed with a standard method.
However, in most cases, the type of bacteria
present in clinical specimens is not known, thus a
nucleic acids extraction method, which is simpler,
fast and can be used with all types of bacteria, is
desired.
Here we made an attempt to use a bead-beating
method as a preparative step prior to standard
bacterial DNA extraction. Bead-beating is a
mechanical disruption method in which beads are
added to the tube containing samples and the
tube is then shaken, causing collisions between
the beads and samples. In recent years, the use of
bead-beating has become more common as a
method for extraction of proteins from plant
tissues８) or for extraction of pathogenic nucleic
acids from yeasts９) and organs10). We tried to
improve the extraction efficiency of nucleic acids
from bacteria by using this bead-beating method
as a preparatory step to physically disrupt cell
walls of Gram-positive bacteria. In this study
Staphylococcus aureus bacteria are used as test
samples since their cell walls are stronger than
those of other Gram-positive bacteria, and results
of the yield and quality of DNA obtained from the
samples under various conditions are reported.
MATERIALS AND METHODS
Bacterial strains
One strain of Staphylococcus aureus 988336B
(laboratory strains) stored in an extreme cold
bath (at −80°C) was scraped with a microtip and
incubated aerobically on staphylococcus medium
No. 100 (Eiken Chemical Co., Ltd.) at 37°C
overnight. Bacteria grown in the culture medium
were then spread across the surface of the
medium and incubated for another 24 hours
under the same conditions. The bacteria with a
bacterial mass of 274.2mg were suspended in 1ml
TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH:
8.0). 200µl of bacterial suspension was dispensed
into each 1.5ml microcentrifuge tube and stored
at −20°C. The emulsions of bacteria stored as
P2
afore-stated were used in each test.
GES method (small scale)
GES method11) was performed with some minor
modifications having been made to scale it down.
① 250µl of GES solution (prepared with 60g
Guanidium Thiocyanate，20ml of 0.5M EDTA
at pH8.0, 5ml of 10% Sarkosyl and distilled
water to 100ml of the solution) was added to
the bacterial emulsion suspended in 50µl TE
buffer and the mixture was left at room
temperature for 5 to 10 minutes and
subsequently cooled on ice.
② After 125µl of 7.5M ammonium acetate was
added and the resulting solution was cooled on
ice for another 10 minutes, 500µl chloroform
was added and mixed gently upside down 50
times. The mixture was then centrifuged, and
the supernatant liquid (the upper layer of
fluid) was decanted into a new tube, added and
mixed with one-half amount of isopropanol,
and DNA was recovered as a pellet by
centrifugation.
③ The pelleted DNA was washed twice with 70%
ethanol and then allowed to dissolve in 200µl
sterile distilled water.
Bead-beating method
Bead-beating disruption was performed between
procedures ① and ② in the GES method
described above and the yields of DNA were
compared. A comparison was made between glass
and zirconia as beads materials used for
disruption. Beads Cell Disrupter Micro Smash
(Tomy Seiko Co., Ltd., Tokyo) was used as a
bead-beating disruption apparatus. Glass or
zirconia beads were placed in 2.0ml sample tubes
(Cat No. 72693 or 72694, Sarstedt, Inc.) and
bacteria suspension in buffer was added to the
tubes, which were then tightly capped and placed
symmetrically in the tube holder of the apparatus
to properly balance them. After the power knob of
the apparatus was fully tightened to secure it in
place, the lid of the disruption chamber was
closed and the samples were bead beaten at the
set speed (rpm) and time (sec).
DNA quantitation
For quantitating DNA, quantitation of double
120124 Optimal Bacterial DNA using bead beating.doc
stranded DNA was calculated from the optical
density absorbance value measured by a
spectrophotometer (ABI) using wavelengths of
260nm and 280nm 12).
RESULTS AND DISCUSSION
In this study, a bead-beating method was
employed for extraction of DNA from
Staphylococcus aureus bacteria which have
stronger cell walls than those of other
Gram-positive species. In the experiment, first,
bead-beating disruption was performed between
procedures ① and ② in the GES method
described previously in MATERIALS AND
METHODS and the DNA yields obtained with
different parameters were compared. Influence of
beads parameters, including material, diameter
and quantity, on DNA yield is shown in Table 1
and influence of disruption speed (speed of
agitating sample tubes) and time on DNA yield is
shown in Table 2. Using the GES method alone,
DNA yield obtained from 13.7mg Staphylococcus
table1 DNA yield
vs bead material, diameter and quantity
Bead
Sample
DNA yield
Material Diameter Quantity
No.
(μg)
(mm)
(mg)
Control
1.1
1
Glass
1.0
50
1.6
2
100
4.5
3
150
10.5
4
200
9.7
5
0.5
50
13.7
6
100
20.0
7
150
19.4
8
200
19.7
9
Zirconia
0.5
50
20.0
10
100
10.9
11
150
6.1
12
200
12.3
at speed: 4,500rpm, time: 30sec
table2 DNA yield
vs speed, time
Agitating condition
DNA yield
Sample
Speed
Time
(μg)
No.
(rpm)
(sec)
13
1.1
14
4,500
30
28.2
15
60
22.6
16
90
12.6
17
120
17.5
18
180
15.5
19
2,400
90
14.2
20
120
8.3
21
180
7.2
at diameter 0.5mm, glass bead 100mg
P3
aureus bacteria was 1.1µg (sample No. 13, Table
2). On the other hand, when performing a
bead-beating procedure, maximum 28.2µg of
DNA yield was obtained from the same amount of
the bacteria (sample No. 14, Table 2).
When comparing glass and zirconia as beads
materials used for disruption, 50mg zirconia
yielded the largest amount of DNA of all
treatment with zirconia beads, however, glass
beads greater than or equal to 100mg yielded
more DNA than 50mg glass beads (Table 1). In all
treatment with glass beads, the 0.5mm diameter
beads yielded more DNA than the 1.0mm
diameter beads (Table 1).It is speculated that this
might be because beads with a shorter diameter
have a larger surface area per unit mass and
therefore provide more frequent contact with
bacteria which have a diameter of approximately
1µm. The results from the treatment with the
0.5mm diameter glass beads showed that there
was not much difference in DNA yields between
the 100mg, 150mg and 200mg beads.
Next, DNA yields obtained at different agitating
speeds and disruption times were compared using
100mg of the 0.5mm diameter glass beads (Table
2). In the studies on agitation speed, DNA yield
obtained from a sample with no bead-beating
treatment was 1.1µg (sample No. 13), which was
lower than the minimum yield of 1.6μg DNA
obtained from a bead-beating sample (sample No.
1). This demonstrates that the use of some
mechanical disruption treatment is considered
more effective for DNA extraction than a
treatment without mechanical disruption. All
samples subjected to disruption at 4,800rpm
yielded more DNA (minimum yield of 15.5μg)
than those at 2,400rpm (maximum yield of
14.2μg), which suggests that agitating the sample
at higher speed exerts more force on bacteria and
facilitates disruption process. In the studies on
disruption time, a short time disruption (30
seconds) demonstrated the highest extraction
efficiency of all samples examined and there was
a tendency for a longer disruption time to reduce
DNA yield. The results from an analysis of DNA
obtained using agarose gel electrophoresis
120124 Optimal Bacterial DNA using bead beating.doc
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clinical specimens cannot be easily found.
Hendolin, et al. extracted DNA in middle ear
effusions containing Haemophilus. influennzae,
Streptococcus penumoniae, Alloiococus otitidis
and Moraxella catarrhalis using a traditional
phenol/ethanol extraction method and extraction
kit（QIAamp DNA mini kit, Qiagen）with the
showed that a DNA cutting occurred more
frequently and the molecular size became smaller
as a disruption time was extended (Figure 1).
Figure 1
Overall, these results demonstrate that
disruption by bead-beating at 4,500rpm for 30
seconds with 100mg of the 0.5mm diameter glass
beads can increase the yield of DNA from
Staphylococcus aureus (by approximately 26
times ) and the use of bead-beating method may
prove useful for bacterial DNA extraction.
Bead-beating method which employs mechanical
disruption of cell walls by beads beating has
facilitated subsequent procedures for DNA
extraction and allowed DNA yield to increase. It
has been reported that a bead-beating method are
useful for protein extraction from tobacco leaves
８), extraction of nucleic acids (DNA and RNA)
from fungi ９ ) (Cryptococcus) and detection of
hepatitis C virus (HCV)-RNA in liver cells10).
The advantage of using a bead-beating method
for bacterial DNA extraction is that there is no
need to change processing methods according to
types of bacteria and the same procedure can
apply to almost all kinds of bacteria, since
enzymatic treatment using a peptidoglycan
nicking enzyme is not required. There is a major
difference
between
Gram-positive
and
Gram-negative bacteria in the structure of their
cell walls and therefore, DNA extraction method
that has the same extraction efficiency in both
Gram-positive and Gram-negative bacteria in
addition of treatment with SDS solution (sodium
dodecyl sulfate-NaOH-chaotropic salt), and
performed multiplex PCR analyses. It is
interesting to note that, although the same
sample was used, phenol/ethanol extraction
resulted in significantly high PCR positive ratio of
Gram-negative bacteria (H. influennzae, M.
catarrhalis), while extraction using the extraction
kit resulted in a significantly high detection rate
of Gram-positive bacteria (S. penumoniae, A.
otitidis ）. In most clinical specimens, types of
bacteria present are unknown and therefore,
employing a bead-beating method is thought to be
beneficial for DNA extraction from clinical
materials14).
The bead-beating method is also faster, as well as
cheaper than enzymatic treatments, allowing for
the speed up of sample testing. It is believed that
this method which permits safe handling of
samples and semiautomatic operation without
depending on the operator's experience can be
highly reproducible. It also allows all the steps
from cell disruption to deproteinization to be
carried out with a single tube and can avoid as
much cross contamination among samples as
possible.
CONCLUSION
In genetic tests and analyses, extraction of DNA
which is then used as a template is the first step
that can have a decisive influence on the test
results. Gram-positive bacteria can be effectively
lysed by chemically break down their cell walls
using a peptidoglycan nicking enzymes. However,
the residues of these enzymes may affect PCR
and therefore, the amount of enzyme should be
minimized if at all possible. There have been no
reports of a method for bacterial lysis that can be
effective against all bacterial cells regardless of
120124 Optimal Bacterial DNA using bead beating.doc
the species, including staphylococci and
streptococci. In this study, we examined the use of
bead-beating method to mechanically disrupt cell
walls. The results showed that of all the methods
performed, a combination of disruption for a short
period of time using glass beads and the GES
method was the most effective one. Another such
method to disrupt cells is ultrasonic disruption
(sonication). Although the use of ultrasonic
disruption was not examined in this study, it may
have the potential to generate infectious aerosols
during the sonication process of specimens, which
can contaminate the surrounding environment
and lead to infection of laboratory workers or
cross contamination among samples. It is thought
that the results of our study on mechanical lysis
of cells and DNA extraction of Staphylococcus
aureus using bead-beating method can apply to
other types of bacteria, therefore, it is expected
that this protocol is useful for direct detection of
bacterial pathogens and causative agents in
clinical specimens that may contain a mixture of
microorganisms.
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