A rapid DNA extraction method for PCR amplification from wetland soils

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE A rapid DNA extraction method for PCR amplification from wetland soils J. Li 1,B.Li 1, Y. Zhou 2,J.Xu 2 and J. Zhao 2 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 , China : received 16 September 2010, revised 1 March 2011 and accepted 15 March 2011 doi: /j x 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 UltraCleanÔ 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. 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; Al 2 (SO 4 ) 3 extraction methods (Dong et al. 2006; Peršoh et al. 2008) use Al 2 (SO 4 ) 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 626 Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology

2 J. Li et al. A rapid DNA extraction method from wetland soils 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 (Hélène et al. 2005), an Al 2 (SO 4 ) 3 extraction method (Peršoh 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 Table 1 Properties of soil sample used in DNA extraction Soil properties W1 W2 W3 W4 Size (lm) and % 1000 lm Æ00 100Æ00 100Æ lm 99Æ89 99Æ67 100Æ00 100Æ lm 96Æ8 90Æ31 91Æ56 96Æ 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 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) (43 38 N, E) and Xilin River (sites W3, W4) (44 08 N, 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 (UltraCleanÔ Soil DNA Isolation kit, Mobio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturers recommendations ( 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 0Æ1 mol l )1 Tris, 0Æ1 mol l )1 Na 4 P 2 O 7, 0Æ1 mol l )1 Na 2 EDTA, 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 Ò 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 (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 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 Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology 627

3 A rapid DNA extraction method from wetland soils J. Li et al. 0 3 g soils 2-ml centrifuge tube + three 2 5-mm-diameter glass beads or 3 0-mm-diameter glass beads 1 ml of a humic-substance-removal solution was added 1 g soil + 2-ml centrifuge tube Homogenized and centrifuged at g for 5 min at ambient temperature, the supernatant was then decanted 1 ml of 0 5 mol l 1 CaCl 2 solution was added, homogenized, and centrifuged at g for 5 min Homogenized for 2 min at maximum speed, centrifuged at 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 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 g for 2 min 900-µl collected supernatant + equal volume of phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the 2-ml centrifuge tube, centrifuged at g for 5 min 800-µl collected supernatant + equal volume of chloroform-isoamyl-alcohol (24 : 1) in the 2-ml centrifuge tube, centrifuged at 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 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 was added, mixed up and down several times and incubated for 30 min at 37 C 200 rpm in a table concentrator, then 200 µl 20% SDS was added and incubated for 1 h, centrifuged at g 900-µl collected supernatant + equal volume of phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the 2-ml centrifuge tube and centrifuged at g 800-µl collected supernatants + equal volume of phenol-chloroform-isoamyl-alcohol (25 : 24 : 1) in the 2-ml centrifuge tube and centrifuged at g 700-µl collected supernatant + equal volume of chloroform-isoamyl-alcohol (24 : 1) in a 1 5-ml centrifuge tube and centrifuged at g 600-µl collected supernatant + equal volume of chloroform-isoamyl-alcohol (24 : 1) in the 1 5-ml centrifuge tube and centrifuged at g 600-µl collected supernatant was incubated for 20 min on ice with 0 6 volume of isopropyl alcohol, centrifuged at g for 5 min The nucleic acids were washed with 70% ethanol and air-dried, 50 µl of TE buffer was added Figure 1 The steps of glass bead calcium chloride SDS method. 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 g The nucleic acids were washed with 70% ethanol and air-dried, followed by addition of 50 µl TE buffer 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 2Æ0 ll of 2Æ5 mmol l )1 dntp, 1Æ0 ll of0æ01 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 of10 buffer (Promega, 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 628 Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology

4 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 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 A 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 72 C, and an additional 10-min cycle at 72 C. 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 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 of10 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 volume of 50 ll using 4Æ0 ll of 2Æ5 10 )3 mol l )1 dntp, 1Æ0 ll of1æ0 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 of10 buffer (Promega) 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 of2æ5 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 of1æ0 10 )9 mol l )1 A643R (5 -TCC CAC TTW GAC CAR GCG GCC ATC CA-3 ) (see Table 2), 5Æ0 ll of10 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 recommendations, ligated into pmd-19t and then transformed into chemically competent Escherichia coli DH-5a (provided 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 ( All sequences were analysed using megablast ( 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 HM HM 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 Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology 629

5 A rapid DNA extraction method from wetland soils J. Li et al. Glass Bead-Calcium Chloride-SDS method M bp 9416 bp 6557 bp 4362 bp 2322 bp 2027 bp 564 bp Ultra Clean Soil DNA Isolation Kit Calcium Chloride-SDS -Enzymatic Method 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) (b) Round 2 Round M 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 UltraCleanÔ 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 16S rdna 27F 1492R AOB amoa A189 2R w + ) ) 1F 2R ww ) AOA amoa A19F A643R ++++ ) ) UltraCleanÔ soil DNA isolation kit 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 Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology

6 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 amoa- 2R provided the most reliable performance in these studies (Rotthauwe et al. 1997). The AmoA gene was amplified GU clone-a-4 HM clone-a-3 HM clone-a-5 HM clone-a-1 HM clone-a-2 HM GU clone-c-6 HM EU FJ clone-b-2 HM clone-b-3 HM clone-b-1 HM HM GQ clone-d-2 HM GU EU GU clone-b-5 HM FJ Nitrosospira-sp.LT2MFa AY clone-c-2 HM clone-c-7 HM FJ Nitrosospira-multiformis DQ AF clone-c-4 HM clone-c-3 HM Nitrosospira-sp.40KI AJ clone-d-1 HM clone-d-4 HM EU clone-d-5 HM clone-d-3 HM AY AB Nitrosomonas-marina AF Nitrosomonas-oligotropha AF Nitrosomonas-ureae AF Cluster 1 Nitrosomonas-eutropha AJ Cluster 2 Cluster Figure 5 Phylogenetic tree based on amoa partial sequences was conducted using MEGA ver. 4Æ0 (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 0Æ05 substitution per nucleotide. Letters in Applied Microbiology 52, ª 2011 The Society for Applied Microbiology 631

7 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 HM HM 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 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. 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