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ORIGINAL ARTICLE
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
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.
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J. Li et al.
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W2
W3
W4
100
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J. Li et al.
03 g soils
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
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J. Li et al.
Sequence
References
Primer synthesis by
27F
1492R
amoA-1F
amoA-2R
A189
A19F
A643R
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
Invitrogen
629
J. Li et al.
Ultra Clean
Calcium
Soil DNA Chloride-SDS
Isolation
-Enzymatic
Kit
Method
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.
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.
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.
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J. Li et al.
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
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clone-A-5|HM481183
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clone-A-1|HM481179
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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
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