Journal of Environmental Sciences 20(2008) 80–87
Purification of total DNA extracted from activated sludge
SHAN Guobin1,2 , JIN Wenbiao3 , Edward K H LAM1 , XING Xinhui2,∗
1. Baolimei Chemical Engineering Co., Ltd., Dongguan 523581, China. E-mail: [email protected]
2. Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
3. Department of Urban and Civil Engineering, Harbin Institute of Technology Shenzhen Graduate School, Shenzhen 518055, China
Received 7 March 2007; revised 19 April 2007; accepted 7 May 2007
Abstract
Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis
treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total
DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for
cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The
combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but
also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of
humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and
polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed
for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in
PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis
(denatured gradient gel electrophoresis) to be possible.
Key words: 16S rDNA; activated sludge; PCR; lysis treatment; DNA purification; wasterwater biotreatment
*Corresponding author. E-mail: [email protected].
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.a
Activated sludge treatment is one of the most popular
biological processes for wastewater treatment (Lee et al.,
2006). The efficiency of this process mainly relies on
the biological activities and settling ability of activated
sludge which consists of diverse microbial communities
organized in aggregates. Analysis of bacterial species and
their diversity in activated sludge is most important for the
characterization of populations and control of wastewater
treatment process (Atlas, 1984). It has been shown that
conventional methods for studying microbial diversity,
such as plating on selective media, are unreliable, because
only a small fraction of the bacterial species present in
the natural habitat can grow on synthetic media (Amann
et al., 1995; Yu and Morrison, 2004). However, molecular
analysis of the natural microbial communities to estimate
bacterial diversity (Wintzingerode et al., 1997; Zhou et
al., 2002) can solve these problems. This approach has
been successfully applied on molecular analysis of clays
(Boivin-Jahns et al., 1996), biofilm (Ahn et al., 2005),
and soil (Ma et al., 2005). The polymerase chain reaction
(PCR) technique is the basis of most molecular methods
for microbial ecology, but this technique is particularly
sensitive to contaminations, such as those caused by humic
substances, proteins and polysaccharides (LaMontagne et
al., 2002). Thus, the extraction and purification of PCRamplifiable DNA from different environmental samples are
two crucial factors ensuring the successful applications of
PCR-based molecular techniques.
In recent years, many methods have been developed
for extracting pure environmental DNA from soils and
sediments; the removal of humic substances and polysaccharides which severely inhibit PCR reactions were
studied (Smalla et al., 1993; Quaiser et al., 2002). Purohit
et al. (2003) reported a pre-processing technique of activated sludge for extraction of PCR-compatible DNA by
using acetone and petroleum ether after pretreatment with
Tween-20.
Many of these protocols have been employed for years;
however, their efficiency and reliability have begun to be
compared only in recent studies. Indirect cell extraction
(in which the cells are separated from the samples prior
to the cell lysis) and direct cell extraction (the microbes
are lysed within the sample) are two basic approaches used
for DNA extraction. Gabor et al. (2003) compared indirect
extraction with direct extraction from diverse environmental samples. Klerks et al. (2006) compared different DNA
extraction kits with respect to DNA extraction efficiency
from soil and compost. Kauffmann et al. (2004) compared
effects of enzymatic treatments with mechanical lysis on
DNA extraction with respect to the yield, purity and
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Introduction
No. 1
Purification of total DNA extracted from activated sludge
degree of shearing. Bourrain et al. (1999) compared two
floc dispersion methods (sonication versus stirring with a
cation exchange resin) and three treatments for cell lysis
(lysozyme, sodium dodecyl sulfate (SDS), sonication and
thermal shock), but they did not investigate the purification
of DNA extracts.
Although some studies on DNA extraction and purification have been reported, the effects of main factors on
the yield and purity of DNA extracted from activated
sludge have not well been analyzed. Here, our study
was to systematically investigate the impacts of most
relevant factors and different purification methods, including extraction buffers, lysis treatment, EDTA (sodium
ethylene-diamine-tetra-acetic acid), SDS, PVP (polyvinyl
pyrrolidone), CTAB (cetyltrimethyl ammonium bromide),
KAc (potassium acetate), PEG-4000 (poly(ethylene glycol)) and phenol/choloride, etc., on the yield and purity of
DNA extracted from activated sludge.
1 Materials and methods
1.1 Activated sludge
Activated sludge samples (5 L) were collected from a
Shenzhen Wastewater Treatment Plant (China) and stored
in plastic containers in dark at 4°C until use (5–6 d). Total
suspended solids value of the sludge was 8300 mg/L. The
value of diluted sludge volume index was 110 ml/g.
1.2 Extraction buffers and phenol/chloroform
mixing 100 ml of tris-EDTA buffer saturated phenol with
96 ml of chloroform.
1.3 Extraction and purification of total DNA
The procedures of extraction and purification of total
DNA from activated sludge are shown in Fig.1. Each
experiment was tested in triplicate. In each test, 0.5 ml
of the sludge sample was used and the DNA extracts
were finally solved into 120 µl of 1×TE (Tris-HCl EDTA)
buffer. The effects of extraction buffers, lysis treatment,
and purification methods were tested on the yield, purity
and diversity of the extracted DNA.
1.4 Evaluation and quantitative analysis of total DNA
DNA concentration was determined spectrophotometrically at 260 nm with a Smart Spec™ plus Spectrophotometer (Bio-Rad, USA). At 1×TE buffer, about one unit
of A260 equals to 50 µg (double chains DNA)/ml. The
purity of DNA was estimated spectrophotometrically by
calculating A260 /A230 and A260 /A280 ratios for evaluation
of humic acid contaminants and protein impurities, respectively (Wilfinger et al., 1997; Sambrook and Russell,
2001). Generally, the A260 /A280 and A260 /A230 ratio of pure
DNA is about 1.8 and more than 2.0, respectively. The
size of DNA fragments isolated was determined by agarose
(1.0%) gel electrophoresis (Sub-cell GT Basic, BIORAD,
Italy) using λ Hind III digested DNA marker or 1 kb
DNA marker (TaKaRa Biotechnology Co., Ltd., China).
The gel was stained with 0.5 µg/ml of ethidium bromide
and photographed under UV light with a camera (Universal
Hood II, Biorad, Italy).
1.5 PCR amplification of 16S rDNA and 16S rDNA V3
region
DNA extracts were used as the template for PCR amplification of the 16S rDNA. The 16S rDNA was amplified,
both with a pair of universal primers (fT1: 8 AGA GTT
TGA TCC TGG CTC AG 27 forward, and rT2: 1406 ACG
GGC GGT GTG TAC AAG 1389 reverse) and a pair of V3
Flow diagram of total DNA extraction and purification from activated sludge.
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Fig. 1
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Two kinds of buffers (TESN and TENP) were used
to extract the total DNA. TESN buffer consists of 100
mmol/L tris-HCl (pH 8.0), 100 mmol/L sodium EDTA
(pH 8.0), 100 mmol/L sodium phosphate (pH 8.0) and
100 mmol/L NaCl. TENP buffer consists of 50 mmol/L
tris-base, 20 mmol/L EDTA, 100 mmol/L NaCl, and 0.01
g/ml polyvinylpyrrolidone. Phenol/chloroform extraction
is an easy way to remove proteins from DNA samples.
The phenol/chloroform solution (pH 8.0) was prepared by
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SHAN Guobin et al.
Vol. 20
region primers (fT3: 325 CCT ACG GGA GGC AGC AG
341 forward, and rT4: 537 ATT ACC GCG GCT GCT GG
518 reverse) corresponding to positions 8–27 and 1389–
1406, 325–341 and 518–537 on the Escherichia coli rrs
sequence (Heuer et al., 1997; Gomes et al., 2001).
The reaction mixture (100 µl) contained 1 × PCR
buffer (10 mmol/L KCl, 8 mmol/L (NH4 )2 SO4 , 10 mmol/L
tris/HCl, pH 9, NP-40), 0.2 mmol/L of each dNTP, 2.5 U
of Taq DNA polymerase, 0.5 µmol/L primer forward and
0.5 µmol/L primer reverse, 20 ng template DNA and 0.2
mmol/L MgCl2 . Cycling was designed with a pre-cycle
(94°C for 5 min) and 30 cycles for amplification (94°C for
1 min, 60°C for 1 min, 72°C for 1 min). PCR amplification
was performed in a MyCycler™ thermal cycler (Bio-Rad,
USA). The electrophoretic migration of PCR products was
performed at 100 V for 30 min on a 1% tris-acetate-EDTA
(TAE) agarose gel using the DNA marker of 1.0 kb or 200
bp.
1.6 DGGE (denatured gradient gel electrophoresis)
analysis
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2.1.1 Extraction buffers, lysis treatments and SDS
Figure 2a shows the effect of extraction buffers on DNA
extraction. The DNA yield extracted by TENP, at about
44.5 mg total DNA/g dried sludge, was about twice of that
by TESN. The A260 /A230 ratio of DNA samples extracted
by TENP was 2.33, while that extracted by TESN was
0.71. This indicated that the humic contaminants were
more efficiently removed by using TENP buffer.
Lysis treatment to disrupt bacterial cells is a primary step
for DNA extraction from activated sludge. The efficiency
of cell disruption affects the yield and purity of the extracted DNA. Five kinds of lysis protocols (sonication (So);
thermal shock (T); and mechanical mill (M); SDS (S);
lysozyme (L)) and combinations of these protocols were
used to disrupt bacterial cells. Lysozyme treatment had to
be tested in TESN buffer because of the high alkalinity
of TENP, while other treatments were tested in TENP
buffer. Since the respective values of A260 , A260 /A230 and
A260 /A280 can be used to evaluate the yield and purity of
DNA, it could be concluded from Fig.2b that the highest
yields were achieved using mechanical mill, sonication and
SDS, while the purest DNA was obtained using SDS and
thermal shock. The concentration of humic substances was
Fig. 3 Agarose gel electrophoresis of DNA fragments extracted using
different lysis treatments. (a) single treatments; (b) combination treatment. (b1) 1 kb marker; (b2) T+S (thermal shock and SDS); (b3) M+S
(mechanical mill and SDS); (b4) So+S (sonication and SDS).
c.
2.1 Effects of process factors on yield and purity of
DNA extracts
the highest after lysozyme treatment due to the absence
of polyvinyl polypyrrolidone (PVPP) in the TESN buffer.
Fig.3a is agarose gel electrophoresis of DNA fragments
extracted using different lysis treatments, which showed
the occurrence of broken DNA when the sludge was
disrupted by sonication, mechanical mill and lysozyme.
However, few small DNA fragments were found when
using thermal shock or 1.5% of SDS.
Considering the DNA yield and purity, using SDS to
disrupt cell was the optimal treatment. Fig.4 shows the microscopic photographs of the activated sludge before being
treated (a) and after being treated with 1.5% SDS treatment
(b). The cells were effectively disrupted. This indicates that
the DNA extraction procedure depends on the ability of the
disruption. The ability of the disruption could be enhanced
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2 Results
Fig. 2 Effects of different buffers (a) and lysis treatments (b) on
A260 /A230 , A260 /A280 and A260 , respectively. So: sonication; T: thermal
shock; M: mechanical mill; S: SDS; L: lysozyme.
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The PCR products of 16S rDNA V3 region were performed by a DGGE apparatus (Dcode™ system, Bio-Rad,
USA). Polyacrylamide gels were composed of 0.06% (v/v)
tetramethylethylenediamine, 0.05% (w/v) ammonium persulfate, 6% acrylamide/methylenebisacrylamide (37.5:1),
0.5×TAE buffer, for 100% denaturing gel mix 40% (v/v)
deionized formamide and 42% (w/v) urea was added.
DGGE was operated in 0.5 × TAE buffer at 60°C and 150
V.
No. 1
Purification of total DNA extracted from activated sludge
Fig. 4
83
Photographs of activated sludge before being treated (a) and after being treated (b) with 1.5% SDS treatment.
by combination of different lysis treatments. In order to
further improve the performance of fragmentation and the
total DNA yield, activated sludge was lysed by combining
other treatment methods with SDS. In this experiment,
SDS was combined with sonication (So+S), mechanical
mill (M+S) and thermal shock (T+S), respectively. TENP
buffer was used as the extraction buffer. Fig.5a shows
that the highest yield was achieved using the combination
of SDS with mechanical mill, while the purest DNA
was obtained using the combination of SDS with thermal
shock. Fig.3b shows that the combination of So+S resulted
in the most shearing of DNA, and T+S resulted in the least
shearing of DNA. Apparently, the yield and the extensive
shearing of total DNA increased by the combination. For
example, compared to the single SDS treatment, A260 was
increased from 25 to 32.6 by combining with sonication,
and from 25 to 34.5 (i.e., 49.9 mg/g) by combining with
mechanical mill. The DNA yield increased about 38%.
However, the combinations had no positive effects on the
purity of total DNA because the change of A260 /A280 value
was very small.
In order to investigate the effect of SDS concentration on
DNA extraction, different SDS concentrations were used
to disrupt the sludge. Fig.5b shows that A260 , A230 and A280
all increased with the increase of SDS concentration, and
started to decrease when the SDS concentration was more
than 2.0%. Accordingly, the efficiency of cell disruption
increased with the increase of SDS concentration. The
maximal yield of DNA extract was about 37.4 mg/g (A260
was 25.9) when the SDS concentration was 1.8%. Also, the
ratio of A260 /A230 was maximal and the ratio of A260 /A280
was the most contiguous to near 1.8. Thus, the purity of
the DNA extract was the highest. Fig.6 shows the size and
characteristics of the DNA fragments extracted by TENP
buffers containing different concentrations of SDS. There
were more shearing DNA fragments using 3.0% of SDS.
The high concentration of SDS (more than 2.0%) tended
to decrease the molecular weight and the purity of DNA.
Thus, the optimal range of SDS concentration was 1.5%–
2.0%.
.a
Fig. 6 Agarose gel electrophoresis of DNA fragments extracted using
different concentrations of SDS. (1) λ Hind III ladder; (2) 3.0%; (3) 2.0%;
(4) 1.5%; (5) 0.6%; (6) 1.2%; (7) 0.4%.
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Fig. 5 Effects of combination treatments (A: themal shock+SDS; B:
mechanical mill+SDS; C: sonication +SDS) (a) and SDS concentrations
(b) on A260 /A230 , A260 /A280 and A260 , respectively.
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2.1.2 EDTA
To study the effect of EDTA on DNA extraction, different EDTA concentrations (10, 20, 40, 60, 80, 100 mmol/L,
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SHAN Guobin et al.
respectively) were examined. Fig.7 shows that all of A260
values increased with the increase of EDTA concentration. There was almost no change in the A260 /A230 ratio
with the increase of EDTA, especially when the EDTA
concentration was higher than 20 mmol/L. A260 /A280 was
most contiguous to 1.8 when EDTA concentration was
20 mmol/L. The increase of EDTA concentration in the
extraction buffer resulted in higher yield but lower purity of
DNA extracts. The optimal range of EDTA concentration
was within 20–40 mmol/L.
2.1.3 PVPP
Figure 8 shows that all of A260 , A230 and A280 decreased
apparently with the increase of PVPP concentration in
TENP buffer. However, the decrease of A260 and A280
values was quicker than that of A230 , which resulted in
the significant decrease of A260 /A230 ratio, especially, when
the PVPP concentration increased up to 5.0% from 3.0%.
According to Fig.8, 1.0% of PVPP concentration was
suitable for the extraction of total DNA from activated
sludge. PVPP at the concentration higher than 1% would
adsorb a large amount of total DNA, and result in lower
DNA yield.
Vol. 20
treatments with CTAB and KAc did not affect A260 /A230
ratio but increased A260 /A280 ratio which was caused by
the decrease of A280 . It was observed that the color of the
crude DNA solutions after CTAB or KAc treatments were
lighter.
2.1.5 Phenol/chloroform (PC) and DNA precipitation
reagents
As shown in Fig.10a, phenol/chloroform (PC) treatment could also enhance the removal of proteins due to
the increase of the A260 /A280 ratio. Following the cell
disruption and pre-purification of the crude DNA, DNA
precipitation is the final step of DNA purification. PEG
(50%), ethanol (95%) and isopropanol were used to precipitate total DNA, respectively. Fig.10b shows the effect of
precipitation reagents on the yield and purity of total DNA.
No significant differences in DNA yield and purity were
2.1.4 CTAB and potassium acetate (KAc)
The effects CTAB and KAc on the purity of DNA
extracted were also investigated. Fig.9 shows the effects
of CTAB and KAc on A260 , A260 /A230 and A260 /A280 . The
Fig. 9 Effects of CTAB and KAc on A260 /A230 , A260 /A280 and A260 ,
respectively. (A) neither; (B) CTAB; (C) CTAB+KAc.
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Fig. 7 Effects of EDTA concentrations on A260 /A230 , A260 /A280 and A260 ,
respectively.
.a
c.
Fig. 10 Effects of phenol/chloroform (PC) extraction (a) and precipitation reagents ((P) PEG; (E) ethanol; (I) isopropanol) (b) on A260 /A230 ,
A260 /A280 and A260 , respectively.
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Fig. 8 Effects of PVPP concentrations on A260 , A230 , A280 , A260 /A230
and A260 /A280 , respectively.
No. 1
Purification of total DNA extracted from activated sludge
observed by ethanol and isopropanol precipitation. All the
A260 /A280 ratios were around 1.8, while the A260 /A230 ratio
was the highest by PEG precipitation.
2.2 PCR amplification and DGGE analysis
Amplification of 16S rDNA and 16S rDNA V3 region
by PCR was succeeded from the DNA extracted from
activated sludge, as demonstrated by the DNA markers of
1.0 kb and 200 bp marker (Fig.11). In order to investigate
the reproducibility of the DNA extraction and purification
procedures, two extracts of total DNA were repeatedly obtained from the same sludge by using the same procedure,
and PCR amplification of 16S rDNA V3 region was then
performed. The two PCR products of 16S rDNA V3 region
were both analyzed by DGGE (Fig.12). The experimental
reproducibility was satisfying.
3 Discussion
Molecular analyses of natural microbial communities
mainly depend on the purity, yield and molecular weight
of the extracted DNA. No DNA fragment was detected
by agarose gel electrophoresis when no lysis treatment
was used (data not shown). Since the total DNA to be
extracted exists inside the cells rather than outside the
cells, lysis treatment is indispensable for DNA extraction from activated sludge. Lysis treatments performed
in our research included physical treatments (ultrasonication, thermal shock and mechanical mill) and chemical
treatments (lysozyme and SDS) and their combinations.
Our results indicated that the highest yields were obtained
using the mechanical mill, sonication and SDS, and the
purest DNA was obtained using SDS and thermal shock.
However, mechanical mill and sonication treatments generally caused severe DNA shearing. Combination of SDS
lysis with other protocols could achieve higher yield of
DNA extracted from activated sludge (Fig.2b and Fig.5a),
but also resulted in severe shearing. The combination of
SDS treatment with thermal shock could achieve much
higher DNA yields and less shearing. Accordingly, the
combination of SDS and thermal shock protocol was
recommended to treat activated sludge for DNA recovery.
Trevors et al. (1992) have also reported that the SDSbased cell lysis protocol can provide the highest DNA yield
compared with other protocols.
In order to obtain DNA products with high purity,
the next step following the disruption treatment is to remove the contaminants from the crude DNA extracts. The
contaminants mainly include humic substances, proteins,
polysaccharides and salts. Among these, humic substances
are high molecular weight materials containing aromatic
rings and nitrogen in cyclic forms or peptide chains formed
by polycondensation (Newman and Theng, 1987). Most
methods for removing humic materials from crude DNA
depend on either different levels of binding of humic
substances and nucleic acids to a polymeric matrix or
fractionation with different sizes (Herrick et al., 1993). The
most widespread techniques are the use of spin columns
packed with various matrices and Sephadex series (Moran
et al., 1993; Tsai and Olson, 1992). However, the use of
spin columns is expensive and complicated, and the purity
is dependent upon the nature of the packing material. Here,
humic substances from crude DNA products were removed
by addition of PVPP in extraction buffer (Fig.2a), which
was a simple, effective and inexpensive method. Fig.9 and
Fig.10a indicate that proteins and polysaccharides were
efficiently removed by precipitation of CTAB and KAc,
and extraction with phenol/chloroform. Then DNA was
further purified by ethanol, isopropanol or PEG precipitation. PEG precipitation resulted in a lower yield but a
higher purity of DNA (Fig.10b). It was consistent with
the result reported by Cullen and Hirsch (1998). Besides,
low EDTA concentration could improve the purity of total
DNA.
The purity will determine the extent to which the
microbial DNA template can be amplified by PCR for
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Fig. 12 DGGE analysis of the PCR products of DNA extracts in the 16S
rDNA V3 region.
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Fig. 11 Agarose gel electrophoresis of the PCR products of the 16S
rDNA and the 16S rDNA V3 region of DNA extracts. (a1) 200 bp marker;
(a2) PCR product of 16S rDNA V3 region of DNA extracts by SDS
disruption; (a3) PCR product of 16S rDNA V3 region of DNA extracts
by disruption of SDS and thermal shock; (b1) 1.0 Kb marker; (b2) PCR
pdroduct of 16S rDNA of DNA extracts by SDS disruption; (b3) PCR
product without DNA template as control; (b4) PCR product of 16S
rDNA of DNA extracts by disruption of SDS and thermal shock.
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SHAN Guobin et al.
Acknowledgements
This work was supported by the China Postdoctoral
Science Foundation (No. 20060390060).
References
Ahn Y, Park E J, Oh Y K, Park S, Webster G, Weightman A J,
2005. Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen production.
FEMS Microbiol Lett, 249(1): 31–38.
Amann R I, Ludwig W, Schleiffer K H, 1995. Phylogenetic
identification and in situ detection of individual microbial
cells without cultivation. Microb Rev, 59: 143–169.
Atlas R M, 1984. Diversity of microbial communities. Adv
Microb Ecol, 7: 1–47.
Boivin-Jahns V, Ruimy R, Bianchi A, Daumas S, Christen R,
1996. Bacterial diversity in a deep subsurface clay environment. Appl Environ Microbiol, 62: 3405–3412.
Bourrain M, Achouak W, Urbain V, Heulin T, 1999. DNA
extraction from activated sludges. Curr Microb, 38: 315–
319.
Cullen D W, Hirsch P R, 1998. Simple and rapid method for
direct extraction of microbial DNA from soil for PCR. Soil
Biol Biochem, 30: 983–993.
Gabor E M, de Vries E J, Janssen D B, 2003. Efficient recovery
of environmental DNA for expression cloning by indirect
extraction methods. FEMS Microb Ecol, 44(2): 153–163.
Gomes N C M, Heuer H, Schönfeld J, Costa R, Mendonça-Hagler
cn
The most reliable procedure of DNA extraction and
purification from activated sludge was investigated. Firstly,
total DNA was directly isolated in TENP buffer by using
1.8% of SDS and thermal shock lysis. Then, the DNA
extract was purified by the subsequent purification steps including CTAB and KAc precipitation, phenol/chloroform
extraction, and isopropanol precipitation. The proposed
protocols could achieve a high yield of about 49.9 mg
(total DNA)/g (volatile suspended solids). The size of
the DNA fragment was about 23 kb, and the extract was
proved to allow the PCR amplifications for the 16S rDNA
and the 16S rDNA V3 regions. The PCR products were
successfully used for DGGE analysis.
c.
4 Conclusions
L, Smalla K, 2001. Bacterial diversity of the rhizosphere
of maize (Zea mays) grown in tropical soil studied by
temperature gradient gel electrophoresis. Plant Soil, 232:
167–180.
Goregues C M, Michotey V D, Bonin P C, 2005. Molecular, biochemical, and physiological approaches for understanding
the ecology of denitrification. Microb Ecol, 49(2): 198–208.
Herrick J B, Madsen E L, Batt C A, Ghiorse W C, 1993.
Polymerase chain reaction amplification of naphthalenecatabolic and 16S rRNA gene sequences from indigenous
sediment bacteria. Appl Environ Microbiol, 59: 687–694.
Heuer H, Krsek M, Baker P, Smalla K, Wellington E M, 1997.
Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic
separation in denaturing gradients. Appl Environ Microbiol,
63: 3233–3241.
Kauffmann I M, Schmitt J, Schmid R D, 2004. DNA isolation
from soil samples for cloning in different hosts. Appl
Microbiol Biotechnol, 64(5): 665–670.
Klerks M M, van Bruggen A H C, Zijlstra C, Donnikov M, 2006.
Comparison of methods of extracting salmonella enterica
serovar enteritidis DNA from environmental substrates and
quantification of organisms by using a general internal
procedural control. Appl Environ Microbiol, 72(6): 3879–
3886.
LaMontagne M G, Michel F C, Holden P A, Reddy C A, 2002.
Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial
community analysis. J Microb Methods, 49(3): 255–264.
Lee T T, Wang F Y, Newell R B, 2006. Advances in distributed
parameter approach to the dynamics and control of activated sludge processes for wastewater treatment. Water Res,
40(5): 853–869.
Ma W K, Siciliano S D, Germida J J, 2005. A PCR-DGGE
method for detecting arbuscular mycorrhizal fungi in cultivated soils. Soil Bio Biochem, 37(9): 1589–1597.
Moran M A, Torsvik V L, Torsvik T, Hodson R E, 1993. Direct
extraction and purification of rRNA for ecological studies.
Appl Environ Microb, 59: 915–918.
Newman B R H, Theng Z K G, 1987. Filip carbon-13 nuclear
magnetic resonance spectroscopic characterisation of humic substances from municipal refuse decomposing in a
landfill. Sci Total Environ, 65: 69–84.
Purohit H J, Kapley A, Moharikar A A, Narde G, 2003. A novel
approach for extraction of PCR-compatible DNA from
activated sludge samples collected from different biological
effluent treatment plants. J Microb Methods, 52: 315–323.
Quaiser A, Ochsenreiter T, Klenk H P, Kletzin A, Treusch A
H, Meurer G et al., 2002. First insight into the genome of
an uncultivated crenarchaeote from soil. Environ Microb,
4(10): 603–611.
Sambrook J, Russell D W, 2001. Spectrophotometry of DNA or
RNA. In: Molecular Cloning. 3rd ed. Cold Spring Harbor,
New York: Cold spring Harbor Laboratory Press. 3: A8.20A8.21.
Smalla K, Cresswell N, Mendonca-Hagler L C, Wolters A, van
Elsas J D, 1993. Rapid DNA extraction protocol from
soil for polymerase chain-reaction-mediated amplification.
J Appl Bacteriol, 74: 78–85.
Trevors J T, Lee H, Cook S, 1992. Direct extraction of DNA from
soil. Microb Releases, 1: 111–115.
Tsai Y L, Olson B H, 1992. Rapid method for separation of
bacterial DNA from humic substances in sediments for
polymerase chain reaction. Appl Environ Microbiol, 58:
.a
the community analysis. The extracted DNA with low
A260 /A230 or unsuitable A260 /A280 ratio failed to be amplified by PCR (data not shown). In this work, total DNA
extracted from activated sludge was successfully used for
PCR amplifications of 16S rDNA, as demonstrated by
the presence of the PCR products of 212 bp and 1.4 kb
(Fig.11). The PCR product of 212 bp was apparently separated into six bands by DGGE (Fig.12), which indicated
that the procedure for DNA extraction and purification was
effective. On the other hand, since the two DGGE lanes
had the same six bands and distribution, the DGGE results
of the DNA extracts obtained by using the procedure was
reproducible. The DNA extract was thus suitable to be
used for molecular studies on the microbial communities
in activated sludge.
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je
sc
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c.
cn
Yu Z, Morrison M, 2004. Comparisons of different hypervariable
regions of rrs genes for use in fingerprinting of microbial
communities by PCR-denaturing gradient gel electrophoresis. Appl Environ Microb, 70: 4800–4806.
Zhou J, Xia B, Treves D S, Wu L Y, Marsh T L, O’Neill R V
et al., 2002. Spatial and resource factors influencing high
microbial diversity in soil. Appl Environ Microb, 68: 326–
334.
.a
2292–2295.
Wilfinger W W, Mackey K, Chomczynski P, 1997. Effect of pH
and ionic strenght on the spectrophotometric assessment of
nucleic acid purity. BioTech, 22: 474–481.
Wintzingerode F, Göbel U B, Stackebrandt E, 1997. Determination of microbial diversity in environmental samples:
pitfalls of PCR based rRNA analysis. FEMS Microb Rev,
21: 213–229.
87
je
sc
No. 1