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Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

A rapid DNA extraction method for PCR amplification from


wetland soils
J. Li1, B. Li1, Y. Zhou2, J. Xu2 and J. Zhao2
1 College of Life Sciences, Inner Mongolia University, Huhhot, China
2 College of Environment and Resources, Inner Mongolia University, Huhhot, China

Keywords
ammonia-oxidizing archaea, ammoniaoxidizing bacteria, calcium chloride, glass
bead, SDS method.
Correspondence
Ji Zhao, College of Environment & Resources,
Inner Mongolia University, Huhhot 010021,
China.
E-mail: ndzj@imu.edu.cn

2010 1633: received 16 September 2010,


revised 1 March 2011 and accepted 15 March
2011
doi:10.1111/j.1472-765X.2011.03047.x

Abstract
Aims: We tested a method of rapid DNA extraction from wetland soil samples
for use in the polymerase chain reaction.
Methods and Results: The glass bead calcium chloride SDS method obtained
in the present study was compared with the calcium chloride SDS enzymatic
extraction method and the UltraClean Soil DNA Isolation Kit. Rapid DNA
extraction could be completed within about two hours without purification
steps.
Conclusions: This study succeeded in establishing a fast soil DNA extraction
protocol that can be applied to various environmental sources that are rich in
humic acid content.
Significance and Impact of the Study: The method provides a technology with
high-quality DNA extraction from soils for testing the diversity of AOB and
AOA.

Introduction
Ammonia oxidation is the fist step in nitrification, a key
process and limiting step in the global nitrogen cycle
(Leininger et al. 2006). Ammonia-oxidizing bacteria
(AOB) and the recently found ammonia-oxidizing organisms belonging to the archaeal domain (AOA) play
important roles in the nitrogen cycle (You et al. 2009).
Ammonia oxidation is now believed to be driven by these
two major microbial groups. AOA have been found in
various habitats including hot thermal springs (Hatzenpichler et al. 2008), oceans (Beman et al. 2008), fresh water
(Santoro et al. 2008) and soil (Tourna et al. 2008). However, they are very difficult to culture outside of these
habitats because of their slow growth rates and their
sensitivity to some organic substances. Up to now, only
AOAs such as Nitrosopumilus maritimu, Nitrosocaldus
yellowstonii, Nitrososphaera gargensis (You et al. 2009)
were obtained. Cultivation-independent methods play
important roles in helping us to understand the diversity
and distribution of these microbes.
Ammonia oxidation-related microbes are low in number
and are hardly detectable using 16S rRNA (Junier et al.
626

2010). Therefore, alternative functional markers such as


specific metabolic-pathway-related key enzymes, e.g., those
involved in ammonia oxidation, have been used for ecological studies (Hermansson and Lindgren 2001). To amplify
the amoA gene from AOA and AOB, an improved
approach based on the dispersal of soils with glass beads is
used to release ammonia-oxidizing microbes that are
strongly adherent on soil colloids or located within the
inner microporosity of soil aggregates (Robe et al. 2003).
Many DNA extraction methods have been reported.
SDS-based methods (Zhou et al. 1996) use CTAB or
PVPP (Juniper et al. 1999) to remove humic substances;
Al2(SO4)3 extraction methods (Dong et al. 2006; Persoh
et al. 2008) use Al2(SO4)3 to remove humic substances
and electroelution methods (Kallmeyer and Smith 2009)
purify DNA directly extracted from marine sediments
with an electroelution apparatus. DNA extraction solutions contain EDTA (Tsai and Olson 1992; Zhou et al.
1996; Miller et al. 1999; Martin-Laurent et al. 2001),
which can combine with the divalent ions. It is very
important to obtain nucleic acids from various environmental samples because DNA techniques allow less biased
access to a greater portion of uncultivable microbes and

2011 The Authors


Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

J. Li et al.

also provide a useful tool for studying the structure and


diversity of microbial communities (Robe et al. 2003).
Many soil DNA extraction methods have been reported,
such as a liquid nitrogen grinding method (Volossiouk
et al. 1995), a microwave-based rupture method (Orsini
and Romano-Spica 2001), an SDS-based method (Zhou
et al. 1996), a bead-beating lysis method (Miller et al.
1999), a rapid freeze-and-thaw method (Tsai and Olson
1992), a cation-exchange method (Jacobsen and Rasmussen 1992), a solvent-based bead-beating method (Chen
et al. 2006), an MS laboratory method (Martin-Laurent
et al. 2001), a Nycodenz gradient separation method
(Hele`ne et al. 2005), an Al2(SO4)3 extraction method
(Persoh et al. 2008), our laboratory-devised calcium chloride SDS enzyme DNA extraction method (Li et al.
2010) and our laboratory-devised glass bead calcium
chloride SDS DNA extraction method, which was
reported in this study. All DNA extraction methods focus
on humic substances. To get high-purity DNA from soil,
these methods generally include a subsequent purification
step, such as Sepharose 4B, Sephadex G-200, Sephadex
G-50 (Jackson et al. 1997) or electroelution (Kallmeyer
and Smith 2009). In our laboratory-devised calcium chloride SDS enzyme DNA extraction method, we also
focused on these substances and obtained a method that
is more efficient at removing humic acids from wetland
soil because it uses a humic-substance-removal solution
combined with calcium chloride solution. However, this
method could only be used for 16S rDNA amplification
and functional gene amplification from certain soils (in
this study). The glass bead calcium chloride SDS DNA
extraction method is based on the humic-substanceremoval technique derived from the calcium chloride SDS enzyme DNA extraction method, and it can
save time when used for functional gene amplification. In
particular, ammonia oxidation-related microbes can be
studied by this method. One commercial DNA purification kit and two laboratory-devised methods, a calcium
chloride SDS enzyme DNA extraction method (Li et al.
2010) and an improved glass bead calcium chloride SDS
method, were used to extract DNA directly from soil. The
amoA gene and 16S rDNA were amplified to estimate the
effectiveness of the different DNA extraction procedures.
Materials and methods
DNA extraction from soil
The physicochemical properties of the four soils used in
this study are presented in Table 1: Microbial biomass
carbon according to the chloroform fumigation extraction
method (Vance et al. 1987), organic carbon content in a
Elementar Liqui TOC Analyzer (Germany), total nitrogen

A rapid DNA extraction method from wetland soils

Table 1 Properties of soil sample used in DNA extraction


Soil properties

W1

W2

W3

W4

Size (lm) and %


1000 lm
500 lm
250 lm
100 lm
50 lm
10 lm
5 lm
2 lm
Organic carbon (g kg)1)
Total nitrogen (g kg)1)
Microbial biomass carbon
(mg kg)1)

100
9989
968
6862
4723
1662
786
1298
7069
2115
98701

10000
9967
9031
5337
3312
1370
649
093
6260
2037
73121

10000
10000
9156
3071
1528
379
098
000
2508
1089
24635

10000
10000
9641
5314
3005
667
222
000
237
17
75610

content with semimicro-Kjeldahl determination (Cole


et al. 1946), grain size distribution in a Microtrac S3500
(Montogomeryville, PA, USA). The experiment was conducted in the Inner Mongolian steppes (sites W1, W2)
(4338N, 11642E) and Xilin River (sites W3, W4)
(4408N, 11708E) in the Inner Mongolia Autonomous
Region, China. Fresh soil samples were sieved (2 mm
mesh) and stored at )20C. DNA was extracted from
four soil samples using a commercial kit (UltraClean
Soil DNA Isolation kit, Mobio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturers recommendations (http://www.mobio. com soil-dna-isolation
ultraclean-soil-dna-isolation-kit.html) and using two procedures developed in our laboratory.
The steps of glass bead calcium chloride SDS method
and the calcium chloride SDS enzyme DNA extraction
method are as follows (see Figs 1 and 2, respectively). A
humic-substance-removal solution containing 01 mol l)1
Tris, 01 mol l)1 Na4P2O7, 01 mol l)1 Na2EDTA, 1%
PVP (w v), 01 mol l)1 NaCl and 005% Triton X-100
(v v), pH 100. DNA extraction buffer containing
01 mol l)1 TrisHCl, 15 mol l)1 NaCl and 1% CTAB,
pH 80. The mixture was homogenized using a VortexGenie 2 (Mobio Laboratories) for glass bead calcium chloride SDS method. If the nucleic acid mix
showed a white precipitate, 500 ll of a sterile, ice-cold
carbonate dissolution mix (043 mol l)1 glacial acetic acid,
043 mol l)1 sodium acetate, and 017 mol l)1 sodium
chloride, pH 46) (Kallmeyer and Smith 2009) was added,
followed by incubation for 20 min on ice with 06 volume
of isopropyl alcohol and centrifugation at 12 000 g for
5 min. Finally, 50 ll of TE buffer was added.
PCR amplification of 16 S rDNA and the amoA gene
To test the quality of the DNA extraction methods, 16S
rDNA and amoA gene amplification was performed on

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Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

627

A rapid DNA extraction method from wetland soils

J. Li et al.

03 g soils

1 g soil + 2-ml centrifuge tube

2-ml centrifuge tube + three 25-mm-diameter


glass beads or 30-mm-diameter glass beads

Homogenized and centrifuged at 12 000 g for 5 min at


ambient temperature, the supernatant was then
decanted

1 ml of a humic-substance-removal solution was


added

1 ml of 05 mol l1 CaCl2 solution was added,


homogenized, and centrifuged at 12 000 g for 5 min

Homogenized for 2 min at maximum speed,


centrifuged at 12 000 g for 2 min at ambient
temperature, followed by decanting of the supernatant

The supernatant was then decanted, 1 ml of 005 mol


l1 sodium oxalate (pH 796) was added,
homogenized and centrifuged at 12 000 g for 5 min
and decanting of the supernatant
700 l of DNA extraction buffer was added, then
homogenized and 100 l of 100 g l1 lysozyme was
added, mixed up and down several times and
incubated for 30 min at 37C 200 rpm in a table
concentrator, then 200 l 20% SDS was added and
incubated for 1 h, centrifuged at 12 000 g

Calcium chloride solution was added (1 ml, 05 mol


l1), homogenized for 2 min at maximum speed,
centrifuged at 12 000 g for 2 min at ambient
temperature, followed by decanting of the supernatant

DNA extraction buffer (800 l) was added


900-l collected supernatant + equal volume of
phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the
2-ml centrifuge tube and centrifuged at 12 000 g

Homogenized for 5 s at maximum speed


200 l 20% SDS was added, mixing up and down

800-l collected supernatants + equal volume of


phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the
2-ml centrifuge tube and centrifuged at 12 000 g

and incubation for 10 min at 65C, centrifuged at


12 000 g for 2 min
900-l collected supernatant + equal volume of
phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the
2-ml centrifuge tube, centrifuged at 12 000 g for 5
min

700-l collected supernatant + equal volume of


chloroform-isoamyl-alcohol (24 : 1) in a 15-ml
centrifuge tube and centrifuged at 12 000 g
600-l collected supernatant + equal volume of
chloroform-isoamyl-alcohol (24 : 1) in the 15-ml
centrifuge tube and centrifuged at 12 000 g

800-l collected supernatant + equal volume of


chloroform-isoamyl-alcohol (24 : 1) in the 2-ml
centrifuge tube, centrifuged at 12 000 g for 5 min

500-l collected supernatant was incubated for 20


min on ice with 06 volume of isopropyl alcohol in the
15-ml centrifuge tube and centrifuged at 12 000 g

600-l collected supernatant was incubated for 20


min on ice with 06 volume of isopropyl alcohol,
centrifuged at 12 000 g for 5 min

The nucleic acids were washed with 70% ethanol and


air-dried, followed by addition of 50 l TE buffer

The nucleic acids were washed with 70% ethanol and


air-dried, 50 l of TE buffer was added
Figure 1 The steps of glass bead calcium chloride SDS method.

Figure 2 The steps of calcium chloride SDS enzyme DNA extraction


method.

DNA obtained directly from the four soils. Three replicates were analysed for the glass bead calcium chloride SDS method. The 16S rDNA was amplified in a
thermocycler from 1 ll of extracted soil DNA template
with a total volume of 25 ll by using 20 ll of
25 mmol l)1 dNTP, 10 ll of 001 mol l)1 27F (5-AGA

GTT TGA TCM TGG CTC AG-3) (see Table 2), 10 ll


of 001 mol l)1 1492R (5-TAC GGH TAC CTT GTT
ACG ACT T-3) (see Table 2), 25 ll of 10 buffer (Promega, Madison, WI), and 02 ll of 5 U ll)1 Taq DNA
polymerase under the following conditions: 5 min at
94C, 30 cycles of 30 s at 94C, 30 s at 55C, and 80 s at

628

2011 The Authors


Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

J. Li et al.

A rapid DNA extraction method from wetland soils

Table 2 List of PCR primers


Name

Sequence

References

Primer synthesis by

27F
1492R
amoA-1F
amoA-2R
A189
A19F
A643R

5-AGA GTT TGA TCM TGG CTC AG-3


5-TAC GGH TAC CTT GTT ACG ACT T-3
5-GGG GTT TCT ACT GGT GGT-3
5-CCC CTC KGS AAA GCC TTC TTC-3
5- GGN GAC TGG GAC TTC TGG-3
5-ATG GTC TGG CTW AGA CG-3
5- TCC CAC TTW GAC CAR GCG GCC ATC CA-3

Martin-Laurent et al. (2001)


Martin-Laurent et al. (2001)
Horz et al. (2004)
Horz et al. (2004)
Horz et al. (2004)
Leininger et al. (2006)
Treusch et al. (2005)

Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen

72C, and an additional 10-min cycle at 72C. The 16S


rDNA PCR products were then separated by electrophoresis on a 1% agarose gel. The AmoA gene of AOB
was amplified using nested PCR. The first round of the
nested PCR amplification from 2 ll of extracted soil
DNA template was conducted in a total volume of 50 ll
by using 40 ll of 25 10)3 mol l)1 dNTP, 10 ll of
10 10)9 mol l)1 A189 (5-GGN GAC TGG GAC TTC
TGG-3) (see Table 2), 10 ll of 10 10)9 mol l)1
amoA-2R (5-CCC CTC KGS AAA GCC TTC TTC-3)
(see Table 2), 50 ll of 10 buffer (Promega) and 04 ll
of 5 U ll)1 Taq under the following conditions: 5 min at
94C, 30 cycles of 40 s at 94C, 40 s at 55C, 40 s at
72C, and an additional 10-min cycle at 72C. The second
round of the nested PCR amplification from 2 ll of
extracted soil DNA template was conducted in a total volume of 50 ll using 40 ll of 25 10)3 mol l)1 dNTP,
10 ll of 10 10)9 mol l)1 amoA-1F (5-GGG GTT TCT
ACT GGT GGT-3) (see Table 2) 10 ll of 10
10)9 mol l)1 amoA-2R (5-CCC CTC KGS AAA GCC
TTC TTC-3) (see Table 2), 50 ll of 10 buffer (Promega) and 04 ll of 5 U ll)1 Taq under the following
conditions: 5 min at 94C, 30 cycles of 20 s at 94C, 20 s
at 55C, 20 s at 72C, and an additional 10-min cycle at
72C. The AmoA gene of AOA was PCR amplified from
2 ll of extracted soil DNA template in a total volume of
50 ll using 80 ll of 25 10)3 mol l)1 dNTP, 10 ll of
10 10)9 mol l)1 A19F (5-ATG GTC TGG CTW AGA
CG-3) (see Table 2), 10 ll of 10 10)9 mol l)1 A643R
(5-TCC CAC TTW GAC CAR GCG GCC ATC CA-3)
(see Table 2), 50 ll of 10 buffer (Promega) and 04 ll
of 5 U ll)1 Taq under the following conditions: 3 min at
95C, 35 cycles of 30 s at 94C, 30 s at 55C, 1 min at
72C, and an additional 10-min cycle at 72C.
Cloning and sequencing
PCR products of the AmoA gene from AOB were purified
using a DNA purification kit (AxyPrep Biosciences,
Hangzhou, China) according to the manufacturers recommendations, ligated into pMD-19T and then transformed
into chemically competent Escherichia coli DH-5a (pro-

vided by the biochemistry laboratory of Inner Mongolia


University, Huhhot, China). Clones were randomly
selected for further analysis. Plasmid DNA was isolated
from individual clones and sequenced by Invitrogen
(Shanghai, China) using M13 forward and reverse primers.
Phylogenetic analysis of AmoA
Gene sequences from AOB were edited, and the vector
sequences were deleted using the CLC Sequence Viewer
5 (http://www.clcbio.com). All sequences were analysed
using megaBlast (http://blast.ncbi.nlm.nih.gov/Blast.cgi?
PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&
PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_
LOC=blasthome) to select the closest reference sequences,
all amoA nucleotide sequences were aligned using
Clustal X software, and an N-J tree (Jukes-Cantor
correction) was constructed using mega software
(Tamura et al. 2007). The bootstrap value was 1000 and
the model was selected using the Kimura-2 parameter.
AmoA gene sequence accession numbers
The sequences identified in this study were submitted to
GenBank under accession numbers HM481179HM481197.
Results
Comparison of the three soil DNA extraction methods
DNA was extracted from four soil samples using three
methods (see the Materials and methods) (Fig. 3). The
two methods devised in our laboratory do not contain
EDTA in the DNA extraction buffer and use a humicsubstance-removal solution and calcium chloride solution
to remove these substances before cell lysis. The glass
bead calcium chloride SDS DNA extraction method
obtains DNA that is about 23 kb in length, and the
procedure can be completed within approximately two
hours. In comparison with the calcium chloride SDS
enzyme DNA extraction method, which needs 4 h (Li
et al. 2010), this method is faster. The glass bead calcium

2011 The Authors


Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

629

A rapid DNA extraction method from wetland soils

J. Li et al.

Ultra Clean
Calcium
Soil DNA Chloride-SDS
Isolation
-Enzymatic
Kit
Method

Glass Bead-Calcium Chloride-SDS method


4 4 4 3 3 3 2 2 2 1 1 1 M

4 3 2 1 4 3 2 1
23130 bp
9416 bp
6557 bp
4362 bp
2322 bp
2027 bp

564 bp

Figure 3 Gel electrophoresis of microbial genomic DNA (three duplicates) from four different soils by three methods.

chloride SDS DNA extraction method saves time and


can be used for PCR amplification (Fig. 4) such as that of
16S rDNA and especially for functional genes (amoA
gene).
Characterization of the new DNA extraction method
The calcium chloride SDS enzyme DNA extraction
method (Li et al. 2010) was efficient in removing humic
substances using the humic-substance-removal solution
combined with the calcium chloride solution, which can
combine with humic substances (Yuan et al. 2000; Davis

et al. 2002; Zhou et al. 2005). These processes are performed only in the first two steps (see the Materials and
methods). As the subsequent steps include lysozyme treatment and heat treatment, they can lead to the release of
humic substances from incompletely degraded animal and
plant cells. For some soils, the humic substances released
during the following steps under lysozyme and heating
treatment cannot be removed. The sensitivity of PCR for
DNA extracted from environmental samples is less than
that for purified genomic DNA. This reduction could be
attributed to humic substances or other interfering compounds present in the soil or sediments (Tsai and Olson
1992). Thus, the calcium chloride SDS enzyme DNA
extraction method could only be used to amplify 16S
rDNA in all soils and the amoA gene from some of the
soils in this study. Thus, the question remains: how to
remove humic substances from incompletely degraded
animal and plant cells? Larger glass beads were chosen to
disperse the cells from the soil particles, allowing most
dead animal and plant cells to release the humic substances into the humic-substance-removal solution and
calcium chloride solution in the first two steps. Therefore,
the humic substances are fully removed, and DNA
obtained by the glass bead calcium chloride SDS DNA
extraction method can be used to amplify functional
genes.

(a)
4

4 4

Round 2
3 3 3 2 2

1 1 1

4 4

Round 1
3 3 2 2

(b)
4
2

1 1

M
2000 bp
1000 bp
750 bp
500 bp
250 bp
100 bp

2000 bp
1000 bp
750 bp
500 bp
250 bp
100 bp

Figure 4 Gel electrophoresis of ammonia-oxidizing bacteria microbial genomic amoA and AOA microbial genomic gene fragment amplification
from four different soils by glass bead calcium chloride SDS method.

Table 3 Results of PCR amplification of 16S


rDNA, amoA gene fragments from ammoniaoxidizing bacteria (AOB) and ammoniaoxidizing archaea (AOA) carried out with the
UltraClean Soil DNA Isolation Kit, calcium
chloride SDS enzymatic and glass beads
calcium chloride SDS method

Gene
fragment
16S rDNA
AOB amoA
AOA amoA

Primer pair

Glass
bead calcium
chloride SDS
method

Calcium
chloride SDS
enzymatic
method

UltraClean
soil DNA
isolation kit

27F 1492R
A189 2Rw
1F 2Rww
A19F A643R

++++
+
++++
++++

++++
)
++
)

++++
)
)
)

The single star represents round 1 of nested PCR, and the double star represents round 2 of nested
PCR. ++++ means that PCR product of the target gene fragment can be obtained from four
different soils by one of the three DNA extraction methods. +++ represents only three different
soils, ++ only two difference soils, + only one soil, ) represents no PCR product of the target gene
fragment can be obtained from four different soils by any one of the DNA extraction methods.

630

2011 The Authors


Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

J. Li et al.

A rapid DNA extraction method from wetland soils

PCR amplification of 16S rDNA and the amoA gene


The results of PCR amplification of 16S rDNA and amoA
are presented in Table 3. The DNA obtained by the three
DNA extraction methods from the four soils was used as a

template to amplify 16S rDNA (Fig. not shown). The specificity of the different primer combinations was tested, and
the primer combination consisting of amoA-1F and amoA2R provided the most reliable performance in these studies
(Rotthauwe et al. 1997). The AmoA gene was amplified

GU931351
clone-A-4|HM481182
72 clone-A-3|HM481181
clone-A-5|HM481183
99
clone-A-1|HM481179
63
clone-A-2|HM481180
61
GU225888
clone-C-6|HM481191
EU515194
Cluster 1
FJ853362
clone-B-2|HM481185
87
clone-B-3|HM481186
clone-B-1|HM481184
HM113507
66
82
GQ143593
87
clone-D-2|HM481194
66
97
GU931355
EU625025
GU931352
84
clone-B-5|HM481187
64
63
FJ890584
Nitrosospira-sp.LT2MFa|AY189145
clone-C-2|HM481188
100 65
clone-C-7|HM481192
100
FJ447346
Nitrosospira-multiformis|DQ228454
AF239880
52
100
clone-C-4|HM481190
99
clone-C-3|HM481189
Nitrosospira-sp.40KI|AJ298687
clone-D-1|HM481193
91
91
99
clone-D-4|HM481196
EU625184
clone-D-5|HM481197
65
clone-D-3|HM481195
100
AY249690
99
89 AB474979
Nitrosomonas-marina|AF272405
Nitrosomonas-oligotropha|AF272406
Nitrosomonas-ureae|AF272403
Nitrosomonas-eutropha|AJ298713
54
59

59
84

Cluster 2

Cluster 3

005
Figure 5 Phylogenetic tree based on amoA partial sequences was conducted using MEGA ver. 40 (Tamura, Dudley, Nei and Kumar 2007). Bootstrap values >50% are shown at each node. Numbers at each branch points indicate the percentage supported by bootstrap based on 1000 replicates. Bar indicates 005 substitution per nucleotide.
2011 The Authors
Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

631

A rapid DNA extraction method from wetland soils

J. Li et al.

using nested PCR because of low abundance of AOB in


our soil samples (Horz et al. 2004). Aliquots of the first
round of PCR products were used as the templates for the
second round of PCR (see the part of Materials and methods). The primer pairs selected were A189 amo-2R and
amo-1F amoA-2R for the first and second rounds of
nested PCR, respectively. The other primer pairs (Junier
et al. 2010) were not tested in this study. The PCR product
from the amoA gene of AOB was 491 bp. AmoA of AOB
was amplified from only one of the four soil samples in
the first round of nested PCR with the glass bead calcium chloride- SDS DNA extraction method (Fig. 4a).
However, the target bands were obtained from all four soil
samples in the second round of nested PCR (Fig. 4a).
AOB were not detected in soil samples W3 or W4 by the
calcium chloride SDS enzyme DNA extraction method.
The abundance and the composition of the indigenous
bacterial community are dependent on the DNA recovery
method used (Martin-Laurent et al. 2001), as clearly demonstrated by our results. The amoA gene of AOA could be
amplified by general PCR from all soils using the primer
pairs A19F A643R (Treusch et al. 2005; Leininger et al.
2006) (Fig. 4b). This finding may indirectly show that the
richness of AOA is higher than that of AOB. AmoA gene
copies from Crenarchaeota (Archaea) are up to 3000-fold
more abundant than bacterial amoA genes (Leininger et al.
2006). AOA amoA genes are more abundant, often as
much as 80 times moreso than AOB amoA genes (Caffrey
et al. 2007). Our results are consistent with the results of
these previous studies.
Phylogenetic relationships
Four clone libraries of the amoA gene were constructed
from the W1, W2, W3 and W4 soil samples. Five randomly selected clones were sequenced. A phylogenetic tree
based on partial amoA sequences was constructed using
mega ver. 4.0 (Tamura, Dudley, Nei, and Kumar 2007). In
this study, 19 clones were obtained from the four soils:
clones A-1 to A-5 belong to the W1 soil sample, clones B-1
to B-3 and B-5 belong to the W2 soil sample, clones C-2
to C-4, C-6 and C-7 belong to the W3 soil sample and
clones D-1 to D-5 belong to the W4 soil sample. These
clones received the accession numbers HM481179
HM481197 from GenBank, respectively (Fig. 5).
The amoA sequences of AOB were divided into three
clusters that were related to Nitrosospira. Nitrosomonas was
not detected in these soil samples. Cluster 1, a Nitrosospira-like group, included five W1 clones, four W2 clones, one
W3 clone and one W4 clone. Cluster 2 included two W3
clones. Cluster 3 included two W3 clones and four W4
clones. The AmoA gene sequences indicated that Nitrosospira of AOB may be the main nitrifiers in these four
632

different soils. Our results are consistent with the findings


of Mohamed et al. (2010).
Conclusion
This study succeeded in establishing a fast soil DNA
extraction protocol that can be applied to various environmental sources that are rich in humic acid content. In
particular, the glass bead calcium chloride SDS method
provides a rapid new approach for studying the diversity
of AOB and ammonia-oxidizing archaea (AOA) in wetland and grassland soils.
Acknowledgements
This work was financially supported by the Key Project of
National Programs for Fundamental Research and Development (2009CB125909). We thank Dr Alexander Buyantuyev for scientific paper writing classes, who is
teaching in success Sino-US for Conservation, Energy and
Sustainability Science. We thank our parents for the support and understanding these years.
References
Beman, J.M., Popp, B.N. and Francis, C.A. (2008) Molecular
and biogeochemical evidence for ammonia oxidation by
marine Crenarchaeota in the Gulf of California. ISME J 2,
429441.
Caffrey, J.M., Bano, N., Kalanetra, K. and Hollibaugh, J.T.
(2007) Ammonia oxidation and ammonia-oxidizing bacteria and archaea from estuaries with differing histories of
hypoxia. ISME J 1, 660662.
Chen, Y.C., Higgins, M.J., Maas, N.A. and Murthy, S.N.
(2006) DNA extraction and Escherichia coli quantification
of anaerobically digested biosolids using the competitive
touchdown PCR method. Water Res 40, 30373044.
Cole, J.E. and Pasks, C.R. (1946) Semi-micro Kjeldahl determination. Ann Chem 18, 6165.
Davis, C.J., Eschenazi, E. and Papadopoulos, K.D. (2002) Combined effects of Ca2+ and humic acid on colliod transport
through porous media. Colloid Polym Sci 280, 5258.
Dong, D.X., Yan, A., Liu, H.M., Zhang, X.H. and Xu, Y.Q.
(2006) Removal of humic substances from soil DNA
using aluminium sulfate. J Microbiol Meth 66, 217222.
Hatzenpichler, R., Lebedeva, E.V., Spieck, E., Stoecker, K.,
Richter, A., Daims, H. and Wagner, M. (2008) A moderately thermophilic ammonia-oxidizing crenarchaeote from
a hot spring. Proc Natl Acad Sci 105, 21342139.
Helene, B., Franck, P., Van, T.V., Nathalie, L., Renaud, N.,
Timothy, M.V. and Pascal, S. (2005) High molecular
weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction. J Microbiol
Methods 62, 111.

2011 The Authors


Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

J. Li et al.

Hermansson, A. and Lindgren, P.E. (2001) Quantification of


ammonia-oxidizing bacteria in arable soil by real-time
PCR. Appl Environ Microbiol 67, 972976.
Horz, H.P., Barbrook, A., Field, C.B. and Bohannan, B.J.M.
(2004) Ammonia- oxidizing bacteria respond to multifactorial global change. Proc Natl Acad Sci 101, 1513615141.
Jackson, C.R., Harper, J.P., Willoughby, D., Roden, E.E. and
Churchill, P.F. (1997) A simple efficient method for the
separation of humic substances and DNA from environmental samples. Appl Environ Microbiol 63, 49934995.
Jacobsen, C.S. and Rasmussen, O.F. (1992) Development and
application of a new method to extract bacterial DNA from
soil based on separation of bacteria from soil with cationexchange resin. Appl Environ Microbiol 58, 24582462.
Junier, P., Molina, V., Dorador, C., Hadas, O., Kim, O.S.,
Junier, T., Witzel, K.P. and Johannes, F.I. (2010) Phylogenetic and functional marker genes to study ammoniaoxidizing microorganisms (AOM) in the environment.
Appl Microbiol Biotechnol 85, 425440.
Juniper, S.K., Cambon, M.A., Lesongeur, F. and Barbier, G.
(1999) Extraction and purification of DNA from organic
rich subsurface sediments. Mar Geol 174, 241247.
Kallmeyer, J. and Smith, D.C. (2009) An improved electroelutionmethod for separation of DNA from humic substances
in marine sediment DNA extracts. FEMS Microbiol Ecol
69, 125131.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol,
G.W., Prosser, J.I., Schuster, S.C. et al. (2006) Archaea predominate among ammonia- oxidizing prokaryotes in soils.
Nature 442, 806809.
Li, J.Y., Zhao, J., Bian, Y., Wu, L.H. and Yu, J.L. (2010) DNA
extraction and removing humic substance from wetland
soil. Microbiol China 37, 11301137.
Martin-Laurent, F., Philippot, L., Hallet, S., Chaussod, R.,
Germon, J.C., Soulas, G. and .Catroux, G. (2001) DNA
extraction from soils: old bias for new microbial diversity
analysis methods. Appl Environ Microbiol 67, 23542359.
Miller, D.N., Bryant, J.E., Madsen, E.L. and Ghiorse, W.C.
(1999) Evaluation and optimization of DNA extraction
and purification procedures for soil and sediment samples.
Appl Environ Microbiol 65, 47154724.
Mohamed, N.M., Saito, K., Tal, Y. and Hill, R.T. (2010) Diversity of aerobic and anaerobic ammonia-oxidizing bacteria
in marine sponges. ISME J 4, 3848.
Orsini, M. and Romano-Spica, V. (2001) A microwave-based
method for nucleic acid isolation from environmental
samples. Lett Appl Microbiol 33, 1720.
Persoh, D., Theuerl, S., Buscot, F. and Rambold, G. (2008)
Towards a universally adaptable method for quantitative

A rapid DNA extraction method from wetland soils

extraction of high-purity nucleic acids from soil. J Microbiol Methods 75, 1924.
Robe, P., Nalin, R., Capellano, C., Vogel, T.M. and Simonet,
P. (2003) Extraction of DNA from soil. Eur J Soil Biol 39,
183190.
Rotthauwe, J.H., Witzel, K.P. and Liesack, W. (1997) The
ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural
ammonia-oxidizing populations. Appl Environ Microbiol
63, 47044712.
Santoro, A.E., Francis, C.A., De-Sieyes, N.R. and Boehm, A.B.
(2008) Shifts in the relative abundance of ammoniaoxidizing bacteria and archaea across physicochemical
gradients in a subterranean estuary. Environ Microbiol 10,
10681079.
Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4:
Molecular Evolutionary Genetic Analysis (MEGA) software
version 4.0. Mol Biol Evol 24, 15961599.
Tourna, M., Freitag, T.E., Nicol, G.W. and Prosser, J.I. (2008)
Growth activity and temperature responses of ammoniaoxidizing archaea and bacteria in soil microcosms. Environ
Microbiol 10, 13571364.
Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C., Klenk,
H.P. and Schleper, C. (2005) Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.
Environ Microbiol 7, 19851995.
Tsai, Y.L. and Olson, B.H. (1992) Detection of low numbers
of bacterial cells in soils and sediments by polymerase
chain reaction. Appl Environ Microbiol 58, 754757.
Vance, E.D., Brookes, P.C. and Jenkinson, D.S. (1987) An
extraction method for measuring soil microbial biomass.
Soil Biol Biochem 19, 703707.
Volossiouk, T., Robb, E.J. and Nazar, R.N. (1995) Direct DNA
extraction for PCR- mediated assays of soil organisms.
Appl Environ Microbiol 61, 39723976.
You, J., Das, A., Dolan, E.M. and Hu, Z.Q. (2009) Ammoniaoxidizing archaea involved in nitrogen removal. Water Res
43, 18011809.
Yuan, G., Theng, B.K.G., Parfitt, R.L. and Percival, H.J. (2000)
Interactions of allophane with humic acid and cations. Eur
J Soil Sci 51, 3541.
Zhou, J.Z., Bruns, M.A. and Tiedje, J.M. (1996) DNA recovery
from soils of diverse composition. Appl Environ Microbiol
62, 316322.
Zhou, P., Yan, H. and Gu, B.H. (2005) Competitive complexation of metal ions with humic substances. Chemosphere 58,
13271337.

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Letters in Applied Microbiology 52, 626633 2011 The Society for Applied Microbiology

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