A Rapid Metagenomic DNA Extraction from Sediments:Potassium Dichromate SDS Method

Jingyu LI

College of Biological Science&Engineering,Beifang Univesity of Nationality,Yinchuan 750021,China

Biodiversity has formed the basis of the Earth’s life support system throughout millions of years of evolution[1].The microbial diversity of the soil has important functions in maintaining the health of wetland ecosystems[2],and estimates of microbial diversity,initially based on the sequencing of clone libraries,have increased greatly in recent years[3].Currently,next-generation sequencing provides genomic data from different habitats worldwide,and the advent of pyrosequencing technology has provided the ability to examine microbial communities at unprecedented levels of coverage and detail[4-6].A recent global survey of bacterial communities from natural environments found that sediment communities may be more phylogenetically diverse than those from any other environment,including soils[7].The excessive input of macronutrient nitrogen results in a change in the trophic status of a given body of water,leading to eutrophication[8],and wetland biogeochemicalprocesses are critical for removing anthropogenic N[9].Indeed,the enrichment of nitrogen in natural ecosystems by inorganic nutrients is occurring at an alarming rate and is perturbing the global N cycle[9].Therefore,the soil microbes involved in anthropogenic nutrient cycling are very important for those systems.

To gain insight into the microbial diversity of a habitat,genomic DNA must be extracted from the environments in which the organisms reside.It is very important to obtain nucleic acids from various environmental samples because DNA-based techniques allow less biased access to a greater proportion of uncultured microbes and also provide a useful tool for studying the structure and diversity of microbial communities[10].Many DNA extraction methods have been reported.SDS-based methods[11]use CTAB or PVPP[12]to remove humic substances,and Al2(SO4)3extraction methods[13-14]use Al2(SO4)3to remove humic substances.A cation-exchange method[15]uses cation exchange to purify the extracted DNA.A glass bead/calcium chloride/SDS DNA extraction method[16]uses a humic substance-removal solution combined with a calcium chloride solution to removal humic substances,and electroelution methods[17]purify the DNA directly extracted from marine sediments using an electroelution apparatus.Clearly,these methods all focus on the removal of humic substances.The present study aims to develop a rapid DNA extraction method for the survey of microbial communities from sediment systems.

Materials and Methods

Sample collection

Three sediment samples were selected at WuLiangSuHai Lake for constructing DNA extraction method.Wuliangsuhai Lake is a large-scale and multifunctional lake.The Lake District(40°47′-41°03′N,108°43′-108°57′E)is located in the western Inner Mongolia Autonomous Region.The elevation of the lake is 1 018.5 m,and the storage capacity is 2.5×108-3×108m3.The existing water area is 333.48 km2,and 80%of the lake is 0.8 m-1.0 m deep.The macrophytes in Wuliangsuhai Lake comprise 11 species from 6 genera and 6 families;Phragmites australisandPotamogeton pectinatusare the dominant species.Wuliangsuhai Lake is primarily supplied by farmland water from the Hetao irrigation district and secondarily by industrial waste water and sewage;an annual total nitrogen flow of 1 088.59 t and total phosphorus flow of 65.75 t enter the lake.Rotting plants accumulate to 9-13 mm per year at the bottom of the lake,accelerating the conversion of Wuliangsuhai Lake to swampland and making it one of the world’s most rapidly filling lakes.The sediment of the lake bottom is approximately 0.1-0.5 m in depth,and the black sediment soils are fetid.The sediment samples were collected from the 0-10 cm layer and stored at-80℃until the DNA was extracted using our novel method.The physicochemical properties of the four samples in this study are presented in Table 1.

Sediments DNA extraction method

DNA was extracted from sediments using a new rapid DNA extraction method following instructions as below.0.3-0.5 g soil was added to 2-ml centrifuge tubes with 3 2.5-mm-diameter glass beads or equal quality of sea sand.Then,600 μl of DNA extraction buffer(0.1 M Tris-HCl,1.5 M Na-Cl,and 1%CTAB,pH 8.0)and 300 μl of potassium dichromate solution(0.05 M)were added and homogenized for 30 s at maximum speed.Finally,100 μl 20%SDS was added,homogenized for 3 min at maximum speed(for glass beads)or for 10 min at maximum speed(for sea sand),and incubated at 65 ℃ for 10 min.100 μl Potassium chloride solution (3 M)was added,mixed several times thoroughly,and centrifuged at 12 000 g for 2 min at ambient temperature.1 000 μl supernatant was added with an equal volume of phenol/chloroform/isoamyl alcohol(25 ∶24 ∶1)and centrifuged at 12 000 g for 5 min.900 μl supernatant was incubated for 10 min on ice with 0.6 volumes of isopropyl alcohol in a 1.5-ml centrifuge tube and centrifuged at 12 000 g for 5 min,and the supernatantwasdecanted.Thenucleic acids were air-dried,and 50 μl ddH2O was added.If the nucleic acid showed a precipitate with color,700 μl precipitation dissolution mix solution(0.43 M glacial acetic acid,0.43 M sodium acetate,and 0.17 M sodium chloride,pH 4.6)[17]was added,incubated for 10 min on ice with 0.6 volumes of isopropyl alcohol,and centrifuged at 12 000 g for 5 min.The nucleic acids were air-dried,and 50 μl ddH2O was added.NOTE:The mixture was homogenized on a Vortex-Genie○R2(Mobio Laboratories Inc).

PCR Detection

To test the quality of the DNA extraction methods,amplification of bacterial 16S rDNA,nosZgene of denitrifying bacteria,pmoAof methanotrophs,nifHof nitrogen-fixing bacteria,amoA ofammonia-oxidizing bacteria and ammonia-oxidizing archaea was performed on DNA obtained directly from the three sediment samples.The 16S rDNA was amplified in a thermocycler from 1 μl of extracted soil DNA template with a total volume of 25 μL by using 2.0 μl of 2.5×10-3M dNTP,1.0 μl of 1×10-5M 27F(5’-AGA GTT TGA TCM TGG CTC AG-3’),1.0 μl of 1×10-5M 1492R(5’-TAC GGH TAC CTT GTT ACG ACT T-3’),2.5 μl of 10×buffer,and 0.2 μl of 5 U/μlTaqDNA polymerase under the following conditions:5 min at 94℃,27 cycles of 30 s at 94℃,30 s at 55℃,and 80 s at 72℃,and an additional 10-min cycle at 72℃.TheAmoAgene of ammoniaoxidizing bacteria was amplified using nested PCR.The first round of the nested PCR amplification from 2 μl of extracted soil DNA template was conducted in a total volume of 50 μl by using 4.0 μl of 2.5×10-3M dNTP,1.0 μl of 1.0×10-5M A189(5’-GGN GAC TGG GAC TTC TGG-3’),1.0 μl of 1.0×10-5M amoA-2R(5’-CCC CTC KGS AAAGCC TTC TTC-3’),5.0 μl of 10 ×buffer,and 0.4 μl of 5 U/μlTaqunder the following conditions:5 min at 94℃,30 cycles of 40 s at 94℃,40 s at 55℃,and 40 s at 72℃,and an additional 10-min cycle at 72℃.The second round of the nested PCR amplification from 2 μl of extracted soil DNA template was conducted in a total volume of 50 μl using 4.0 μl of 2.5×10-3M dNTP,1.0 μl of 1.0×10-5M amoA-1F(5’-GGG GTT TCT ACT GGT GGT-3’),1.0 μl of 1.0×10-5M amoA-2R,5.0 μl of 10×buffer,and 0.4 μl of 5 U/μlTaqunder the following conditions:5 min at 94℃,15 cycles of 20 s at 94℃,20 s at 55℃,and 20 s at 72℃,and an additional 10-min cycle at 72℃.TheAmoAgene of ammonia-oxidizing archaea was PCR-amplified from 2 μl of extracted soil DNA template in a total volume of 50 μl using 4.0 μl of 2.5×10-3M dNTP,1.0 μl of 1.0×10-5M A19F(5’-ATG GTC TGG CTW AGA CG-3’),1.0 μl of 1.0×10-5M A643R(5’-TCC CAC TTW GAC CAR GCG GCC ATC CA-3’),5.0 μl of 10×buffer and 0.4 μl of 5 U/μlTaqunder the following conditions:3 min at 95℃,35 cycles of 30 s at 94℃,30 s at 55℃,and 1 min at 72℃,and an additional 10-min cycle at 72℃.TheNosZgene of denitrifying bacteria was amplified using nested PCR.The first round of the nested PCR amplification from 1 μl of extracted soil DNA template was conducted in a total volume of 25 μl by using 2.0 μl of 2.5×10-3mol/L dNTP,0.5 μl of 1.0×10-5mol/L nosZ-F(5’-CGY TGT TCM TCG ACA GCC AG-3’),0.5 μl of 1.0×10-5mol/L nosZ-R(5’-CAT GTG CAG NGC RTG GCA GAA-3’),2.5 μl of 10×buffer,and 0.125 μl of 5 U/μl Taq under the following conditions:2 min at 94℃,25 cycles of 30 s at 94℃,60 s at 50℃,and 60 s at 72℃,and an additional 10-min cycle at 72℃.The second round of the nested PCR amplification from 1 μl of extracted soil DNA template was conducted in a total volume of 25 μl using 2.0 μl of 2.5 ×10-3mol/L dNTP,0.5 μl of 1.0×10-5mol/L nosZ-F,0.5 μl of 1.0×10-5mol/L nosZ 1622R(5’-CGS ACC TTS TTG CCS TYG CG-3’),2.5 μl of 10×buffer,and 0.125 μl of 5 U/μl Taq under the following conditions:2 min at 94℃,35 cycles of 30 s at 94℃,30 s at 58℃,and 60 s at 72℃,and an additional 10-min cycle at 72℃.ThepmoAgene of methanotrophs was amplified using nested PCR.The first round of the nested PCR amplification from 1 μl of extracted soil DNA template was conducted in a total volume of 25 μl by using 2.0 μl of 2.5×10-3mol/L dNTP,1.0 μl of 1.0 ×10-5mol/L A189,1.0 μl of 1.0 ×10-5mol/L A682(5’-GAA SGC NGA GAA GAA SGC-3’),2.5 μl of 10×buffer,and 0.2 μl of 5 U/μlTaqunder the following conditions:3 min at 95℃,35 cycles of 60 s at 95℃,60 s at 62℃,and 60 s at 72℃,and an additional 10-min cycle at 72℃.The second round of the nested PCR amplification from 1 μl of extracted soil DNA template was conducted in a total volume of 25 μl using 2.0 μl of 2.5 ×10-3mol/L dNTP,1.0 μl of 1.0×10-5mol/L A189,1.0 μl of 1.0×10-5mol/L mb661(5’-CCG GMG CAA CGT CYT TAC C-3’),2.5 μl of 10×buffer,and 0.2 μl of 5 U/μlTaqunder the following conditions:3 min at 95℃,30 cycles of 60 s at 95℃,60 s at 55℃,and 40 s at 72℃,and an additional 10-min cycle at 72℃.ThenifHgene of nitrogen-fixing bacteria was PCR-amplified from 2 μl of extracted soil DNA template in a total volume of 50 μl using 4.0 μl of 2.5 ×10-3mol/L dNTP,1.0 μl of 1.0×10-5mol/L PolF(5’-TGC GAY CCS AAR GCB GAC TC-3’),1.0 μl of 1.0×10-5mol/L PolR(5’-ATS GCC ATC ATY TCR CCG GA-3’),5.0 μl of 10×buffer and 0.25 μl of 5 U/μlTaqunder the following conditions:2 min at 94℃,30 cycles of 1 min at 94℃,1 min at 55℃,and 2 min at 72℃,and an additional 10-min cycle at 72℃.

Table 1 Properties of sediment samples used for constructing DNA extraction method

Results and Discussion

Our DNA extraction method utilized potassium dichromate solution in the initial step and was efficient at completely removing the interfering substances,enabling DNA extraction from black and fetid sediment soils.Moreover,we improved the speed of extraction to approximately 1 h.In our method,the steps of humic substance removal and DNA extraction are performed simultaneously,and the nucleic acids that are obtained with this method do not need to be washed with 70%ethanol.Because the steps of adding an equal volume of isoamylalcohol(24∶1)and washing with 70%ethanolare unnecessary in our method,the nucleic acids are dissolved directly in sterile water for effective molecular analyses.The results of the PCR amplification of the 16S rDNA,amoAof ammonia-oxidizing bacteria and ammonia-oxidizing archaea,nosZof denitrifying bacteria,pmoAof methanotrophs,andnifHof nitrogen-fixing bacteria are presented in Fig.1.The DNA that was obtained in parallel from the three sediment soils usingourextractionmethodswas used as the template to amplify these genes.nosZ,pmoA,nifH,amoAand the 16S rDNA were successfully amplified,demonstrating the effectiveness of the DNA extraction procedure.

This study succeeded in estab-lishing a rapid DNA extraction protocol consisting of one step of rapid DNA extraction with humus removal from black and fetid sediments that are rich in humic acid content.Although there is an improved electroelution method for the separation of DNA from humic substances in marine sediment extracts,this technique requires a special electroelution apparatus to purify the crude DNA extracts[17].Single minicolumn,double minicolumn,gel plus minicolumn and gel plus centrifugal concentrator methods have been used in SDS-based DNA extraction methods[11]for the purification of crude DNA extracts.A glass bead/calcium chloride/SDS DNA extraction method[16]has also been successfully used for the amplification ofamoAgenes of ammonia-oxidizing bacteria and ammonia-oxidizing archaea from grassland soils and wetland soils[18]and the amplification of thenosZgene of denitrifying bacteria and pmoA of methanotrophs from wetland soils and grassland soils[19].Although microbial DNA of sediment soils in some environments (such as the soil samples described here)can be extracted by a more recent method[16],which is not suitable for the PCR amplification of functional genes.Accordingly,to address these issues,we used potassium dichromate solution in the extraction procedure instead of a humic-substance removal solution combined with calcium chloride solution[16].Compared with previous methods,this novel procedure does not require post-treatment for crude DNA extracts,which can be used directly foreffective molecular analyses.Some commonly used commercial soil DNA isolation kits are inefficient for some types of soils.For instance,wastewater treatment systems in which the DNA was obtained using the Power soil DNA isolation kit(Mo Bio,Carlsbad,CA)was not suitable for the PCR amplification ofamoAgenes[20],and the DNA extracted from wetland soils using the UltraClean Soil DNA Isolation kit(Mo Bio,Carlsbad,CA)was also not suitable for amoA gene PCR amplification[16].The potassium dichromate solution used in this study for the first time is very suitable for the pre-treatment of black and fetid sediment soils for DNA extraction for use in PCR amplification.

In summary,the DNA that was obtained using our extraction method can be used to amplify 16S rDNA and nitrogen-related and carbon-related genes from sediment soils.

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