A Rapid DNA Extraction Method for PCR Amplification From Wetland Soils

ORIGINAL ARTICLE

A rapid DNA extraction method for PCR amplification fromwetland soilsJ. 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

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 organ-

isms 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 (Hatzenp-

ichler et al. 2008), oceans (Beman et al. 2008), fresh water

(Santoro et al. 2008) and soil (Tourna et al. 2008). How-

ever, 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.

2010). Therefore, alternative functional markers such as

specific metabolic-pathway-related key enzymes, e.g., those

involved in ammonia oxidation, have been used for ecolog-

ical 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 solu-

tions 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 environ-

mental samples because DNA techniques allow less biased

access to a greater portion of uncultivable microbes and

Keywords

ammonia-oxidizing archaea, ammonia-

oxidizing bacteria, calcium chloride, glass

bead, SDS method.

Correspondence

Ji Zhao, College of Environment & Resources,

Inner Mongolia University, Huhhot 010021,

China.

E-mail: [email protected]

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.

Letters in Applied Microbiology ISSN 0266-8254

626 Letters in Applied Microbiology 52, 626–633 ª 2011 The Society for Applied Microbiology

ª 2011 The Authors

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 Rasmus-

sen 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

(Helene et al. 2005), an Al2(SO4)3 extraction method

(Persoh et al. 2008), our laboratory-devised calcium chlo-

ride ⁄ 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 chlo-

ride ⁄ 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-substance-

removal technique derived from the calcium chlo-

ride ⁄ 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 purifica-

tion 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

content with semimicro-Kjeldahl determination (Cole

et al. 1946), grain size distribution in a Microtrac S3500

(Montogomeryville, PA, USA). The experiment was con-

ducted in the Inner Mongolian steppes (sites W1, W2)

(43�38¢N, 116�42¢E) and Xilin River (sites W3, W4)

(44�08¢N, 117�08¢E) in the Inner Mongolia Autonomous

Region, China. Fresh soil samples were sieved (2 mm

mesh) and stored at )20�C. DNA was extracted from

four soil samples using a commercial kit (UltraClean�Soil DNA Isolation kit, Mobio Laboratories Inc., Carls-

bad, CA, USA) according to the manufacturers’ recom-

mendations (http://www.mobio. com ⁄ soil-dna-isolation ⁄ultraclean-soil-dna-isolation-kit.html) and using two pro-

cedures 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 0Æ1 mol l)1

Tris, 0Æ1 mol l)1 Na4P2O7, 0Æ1 mol l)1 Na2EDTA, 1%

PVP (w ⁄ v), 0Æ1 mol l)1 NaCl and 0Æ05% Triton X-100

(v ⁄ v), pH 10Æ0. DNA extraction buffer containing

0Æ1 mol l)1 Tris–HCl, 1Æ5 mol l)1 NaCl and 1% CTAB,

pH 8Æ0. The mixture was homogenized using a Vortex-

Genie� 2 (Mobio Laboratories) for glass bead ⁄ cal-

cium ⁄ chloride ⁄ SDS method. If the nucleic acid mix

showed a white precipitate, 500 ll of a sterile, ice-cold

carbonate dissolution mix (0Æ43 mol l)1 glacial acetic acid,

0Æ43 mol l)1 sodium acetate, and 0Æ17 mol l)1 sodium

chloride, pH 4Æ6) (Kallmeyer and Smith 2009) was added,

followed by incubation for 20 min on ice with 0Æ6 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

Table 1 Properties of soil sample used in DNA extraction

Soil properties W1 W2 W3 W4

Size (lm) and %

1000 lm 100 100Æ00 100Æ00 100Æ00

500 lm 99Æ89 99Æ67 100Æ00 100Æ00

250 lm 96Æ8 90Æ31 91Æ56 96Æ41

100 lm 68Æ62 53Æ37 30Æ71 53Æ14

50 lm 47Æ23 33Æ12 15Æ28 30Æ05

10 lm 16Æ62 13Æ70 3Æ79 6Æ67

5 lm 7Æ86 6Æ49 0Æ98 2Æ22

2 lm 1Æ298 0Æ93 0Æ00 0Æ00

Organic carbon (g kg)1) 70Æ69 62Æ60 25Æ08 2Æ37

Total nitrogen (g kg)1) 2Æ115 2Æ037 1Æ089 1Æ7

Microbial biomass carbon

(mg kg)1)

987Æ01 731Æ21 246Æ35 756Æ10

J. Li et al. A rapid DNA extraction method from wetland soils

ª 2011 The Authors

Letters in Applied Microbiology 52, 626–633 ª 2011 The Society for Applied Microbiology 627

DNA obtained directly from the four soils. Three repli-

cates were analysed for the glass bead ⁄ calcium ⁄ chlo-

ride ⁄ 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 2Æ0 ll of

2Æ5 mmol l)1 dNTP, 1Æ0 ll of 0Æ01 mol l)1 27F (5¢-AGA

GTT TGA TCM TGG CTC AG-3¢) (see Table 2), 1Æ0 ll

of 0Æ01 mol l)1 1492R (5¢-TAC GGH TAC CTT GTT

ACG ACT T-3¢) (see Table 2), 2Æ5 ll of 10· buffer (Pro-

mega, Madison, WI), and 0Æ2 ll of 5 U ll)1 Taq DNA

polymerase under the following conditions: 5 min at

94�C, 30 cycles of 30 s at 94�C, 30 s at 55�C, and 80 s at

The nucleic acids were washed with 70% ethanol and

air-dried, 50 µl of TE buffer was added

600-µl collected supernatant was incubated for 20min on ice with 0·6 volume of isopropyl alcohol,

centrifuged at 12 000 g for 5 min

800-µl collected supernatant + equal volume ofchloroform-isoamyl-alcohol (24 : 1) in the 2-ml

centrifuge tube, centrifuged at 12 000 g for 5 min

900-µl collected supernatant + equal volume ofphenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the

2-ml centrifuge tube, centrifuged at 12 000 g for 5min

0·3 g soils

2-ml centrifuge tube + three 2·5-mm-diameterglass beads or 3·0-mm-diameter glass beads

1 ml of a humic-substance-removal solution wasadded

Homogenized for 2 min at maximum speed, centrifuged at 12 000 g for 2 min at ambient

temperature, followed by decanting of the supernatant

Calcium chloride solution was added (1 ml, 0·5 mol

l–1), 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 µl) was added

Homogenized for 5 s at maximum speed

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

and incubation for 10 min at 65°C, centrifuged at

12 000 g for 2 min

Figure 1 The steps of glass bead ⁄ calcium ⁄ chloride ⁄ SDS method.

The nucleic acids were washed with 70% ethanol and

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

500-µl collected supernatant was incubated for 20 min on ice with 0·6 volume of isopropyl alcohol in the

1·5-ml centrifuge tube and centrifuged at 12 000 g

600-µl collected supernatant + equal volume ofchloroform-isoamyl-alcohol (24 : 1) in the 1·5-ml

centrifuge tube and centrifuged at 12 000 g

800-µl collected supernatants + equal volume ofphenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the

2-ml centrifuge tube and centrifuged at 12 000 g

1 g soil + 2-ml centrifuge tube

Homogenized and centrifuged at 12 000 g for 5 min atambient temperature, the supernatant was then

decanted

1 ml of 0·5 mol l–1 CaCl2 solution was added,homogenized, and centrifuged at 12 000 g for 5 min

The supernatant was then decanted, 1 ml of 0·05 mol

l–1 sodium oxalate (pH 7·96) was added,homogenized and centrifuged at 12 000 g for 5 min

and decanting of the supernatant

700 µl of DNA extraction buffer was added, then

homogenized and 100 µl of 100 g l–1 lysozyme wasadded, mixed up and down several times and

incubated for 30 min at 37°C 200 rpm in a tableconcentrator, then 200 µl 20% SDS was added and

incubated for 1 h, centrifuged at 12 000 g

900-µl collected supernatant + equal volume of phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the

2-ml centrifuge tube and centrifuged at 12 000 g

700-µl collected supernatant + equal volume ofchloroform-isoamyl-alcohol (24 : 1) in a 1·5-ml

centrifuge tube and centrifuged at 12 000 g

Figure 2 The steps of calcium chloride ⁄ SDS ⁄ enzyme DNA extraction

method.

A rapid DNA extraction method from wetland soils J. Li et al.


ª 2011 The Authors

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

rDNA PCR products were then separated by electro-

phoresis 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 4Æ0 ll of 2Æ5 · 10)3 mol l)1 dNTP, 1Æ0 ll of

1Æ0 · 10)9 mol l)1 A189 (5¢-GGN GAC TGG GAC TTC

TGG-3¢) (see Table 2), 1Æ0 ll of 1Æ0 · 10)9 mol l)1

amoA-2R (5¢-CCC CTC KGS AAA GCC TTC TTC-3¢)(see Table 2), 5Æ0 ll of 10 · buffer (Promega) and 0Æ4 ll

of 5 U ll)1 Taq under the following conditions: 5 min at

94�C, 30 cycles of 40 s at 94�C, 40 s at 55�C, 40 s at

72�C, and an additional 10-min cycle at 72�C. The second

round of the nested PCR amplification from 2 ll of

extracted soil DNA template was conducted in a total vol-

ume of 50 ll using 4Æ0 ll of 2Æ5 · 10)3 mol l)1 dNTP,

1Æ0 ll of 1Æ0 · 10)9 mol l)1 amoA-1F (5¢-GGG GTT TCT

ACT GGT GGT-3¢) (see Table 2) 1Æ0 ll of 1Æ0 ·10)9 mol l)1 amoA-2R (5¢-CCC CTC KGS AAA GCC

TTC TTC-3¢) (see Table 2), 5Æ0 ll of 10 · buffer (Pro-

mega) and 0Æ4 ll of 5 U ll)1 Taq under the following

conditions: 5 min at 94�C, 30 cycles of 20 s at 94�C, 20 s

at 55�C, 20 s at 72�C, and an additional 10-min cycle at

72�C. The AmoA gene of AOA was PCR amplified from

2 ll of extracted soil DNA template in a total volume of

50 ll using 8Æ0 ll of 2Æ5 · 10)3 mol l)1 dNTP, 1Æ0 ll of

1Æ0 · 10)9 mol l)1 A19F (5¢-ATG GTC TGG CTW AGA

CG-3¢) (see Table 2), 1Æ0 ll of 1Æ0 · 10)9 mol l)1 A643R

(5¢-TCC CAC TTW GAC CAR GCG GCC ATC CA-3¢)(see Table 2), 5Æ0 ll of 10 · buffer (Promega) and 0Æ4 ll

of 5 U ll)1 Taq under the following conditions: 3 min at

95�C, 35 cycles of 30 s at 94�C, 30 s at 55�C, 1 min at

72�C, and an additional 10-min cycle at 72�C.

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’ recom-

mendations, 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 HM481179–HM481197.

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 humic-

substance-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

Table 2 List of PCR primers

Name Sequence References Primer synthesis by

27F 5¢-AGA GTT TGA TCM TGG CTC AG-3¢ Martin-Laurent et al. (2001) Invitrogen

1492R 5¢-TAC GGH TAC CTT GTT ACG ACT T-3¢ Martin-Laurent et al. (2001) Invitrogen

amoA-1F 5¢-GGG GTT TCT ACT GGT GGT-3¢ Horz et al. (2004) Invitrogen

amoA-2R 5¢-CCC CTC KGS AAA GCC TTC TTC-3¢ Horz et al. (2004) Invitrogen

A189 5¢- GGN GAC TGG GAC TTC TGG-3¢ Horz et al. (2004) Invitrogen

A19F 5¢-ATG GTC TGG CTW AGA CG-3¢ Leininger et al. (2006) Invitrogen

A643R 5¢- TCC CAC TTW GAC CAR GCG GCC ATC CA-3¢ Treusch et al. (2005) Invitrogen


ª 2011 The Authors


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 per-

formed only in the first two steps (see the Materials and

methods). As the subsequent steps include lysozyme treat-

ment 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 com-

pounds 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 sub-

stances 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) (b)

2000 bp

M111222333444111222333444Round 2 Round 1 4 3 2 1 M

1000 bp750 bp500 bp250 bp100 bp

2000 bp1000 bp750 bp500 bp250 bp100 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.

Glass Bead-Calcium Chloride-SDS method

Ultra CleanSoil DNAIsolation

Kit

CalciumChloride-SDS

-EnzymaticMethod4 4 4 3 3 3 2 2 2 1 1 1 M

4 3 2 1 4 3 2 1

23130 bp9416 bp6557 bp4362 bp2322 bp2027 bp

564 bp

Figure 3 Gel electrophoresis of microbial genomic DNA (three dupli-

cates) from four different soils by three methods.

Table 3 Results of PCR amplification of 16S

rDNA, amoA gene fragments from ammonia-

oxidizing bacteria (AOB) and ammonia-

oxidizing archaea (AOA) carried out with the

UltraClean� Soil DNA Isolation Kit, calcium

chloride ⁄ SDS ⁄ enzymatic and glass beads ⁄calcium chloride ⁄ SDS method

Gene

fragment Primer pair

Glass

bead ⁄ calcium

chloride ⁄ SDS

method

Calcium

chloride ⁄ SDS ⁄enzymatic

method

UltraClean�soil DNA

isolation kit

16S rDNA 27F ⁄ 1492R ++++ ++++ ++++

AOB amoA A189 ⁄ 2Rw + ) )1F ⁄ 2Rww ++++ ++ )

AOA amoA 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.



ª 2011 The Authors

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 spec-

ificity of the different primer combinations was tested, and

the primer combination consisting of amoA-1F and amoA-

2R provided the most reliable performance in these studies

(Rotthauwe et al. 1997). The AmoA gene was amplified

GU931351

clone-A-4|HM481182

clone-A-3|HM481181

clone-A-5|HM481183

clone-A-1|HM481179

clone-A-2|HM481180

GU225888

clone-C-6|HM481191

EU515194

FJ853362

clone-B-2|HM481185

clone-B-3|HM481186

clone-B-1|HM481184

HM113507

GQ143593

clone-D-2|HM481194

GU931355

EU625025

GU931352

clone-B-5|HM481187

FJ890584

Nitrosospira-sp.LT2MFa|AY189145

clone-C-2|HM481188

clone-C-7|HM481192

FJ447346

Nitrosospira-multiformis|DQ228454

AF239880

clone-C-4|HM481190

clone-C-3|HM481189

Nitrosospira-sp.40KI|AJ298687

clone-D-1|HM481193

clone-D-4|HM481196

EU625184

clone-D-5|HM481197

clone-D-3|HM481195

AY249690

AB474979

Nitrosomonas-marina|AF272405

Nitrosomonas-oligotropha|AF272406

Nitrosomonas-ureae|AF272403

Nitrosomonas-eutropha|AJ298713

100

52100

8999

100

63

5459

72

99

9199

99

97

65

91

87

6687

6364

84

66

61

82

65100

8459

0·05

Cluster 1

Cluster 2

Cluster 3

Figure 5 Phylogenetic tree based on amoA partial sequences was conducted using MEGA ver. 4Æ0 (Tamura, Dudley, Nei and Kumar 2007). Boot-

strap values >50% are shown at each node. Numbers at each branch points indicate the percentage supported by bootstrap based on 1000 repli-

cates. Bar indicates 0Æ05 substitution per nucleotide.


ª 2011 The Authors


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 meth-

ods). 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 ⁄ cal-

cium ⁄ 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 dem-

onstrated 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 ran-

domly 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 Nitrosospir-

a-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 Nitroso-

spira of AOB may be the main nitrifiers in these four

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 envi-

ronmental 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 wet-

land and grassland soils.

Acknowledgements

This work was financially supported by the Key Project of

National Programs for Fundamental Research and Devel-

opment (2009CB125909). We thank Dr Alexander Buy-

antuyev for scientific paper writing classes, who is

teaching in success Sino-US for Conservation, Energy and

Sustainability Science. We thank our parents for the sup-

port and understanding these years.

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A Rapid DNA Extraction Method for PCR Amplification From Wetland Soils

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