Research Article An Improved Method for Soil DNA Extraction ...Research Article An Improved Method...

Research ArticleAn Improved Method for Soil DNA Extraction to Studythe Microbial Assortment within Rhizospheric Region

Faria Fatima1 Neelam Pathak1 and Smita Rastogi Verma2

1 Department of Biosciences Integral University Lucknow 226026 India2Department of Biotechnology Delhi Technological University Delhi 110042 India

Correspondence should be addressed to Smita Rastogi Verma srsmitarastogigmailcom

Received 18 May 2014 Revised 10 August 2014 Accepted 19 August 2014 Published 15 September 2014

Academic Editor Malayannan B Subramaniam

Copyright copy 2014 Faria Fatima et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil a multifacetedenvironment that is a major pool for microbial genetic diversityThe soil DNA extraction procedures usually suffer from twomajorproblems namely inappropriate rupturing of cells and contamination with humic substances In the present study five protocolsfor single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120mM phosphatebuffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time withno specific equipment requirement Furthermore DNA purity and yield were also improved which allowed the exploitation ofgenetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNAThe effectivenessofmethods was analyzed using random amplification of polymorphic DNAThe banding patterns revealed that both the abundanceand the composition of indigenous microbial community depend on the DNA recovery method

1 Introduction

The biodiversity of microbes within soil is significant forthe maintenance of healthy soil because these microbes areinvolved in many vital functions like crucial cycles of CN P formation of soil toxin removal and so on Previ-ously studies on the development of microbial communitiesrequired the isolation of these microbes from soil sample byculture dependent techniques followed by a series test forphenotypic evaluation and their identification However themicrobial diversity studies conducted in soil have been biasedessentially due to the unculturability of many microbesSpecific media which are used to culture microbes areselective in nature and only subpopulations of microbes fromenvironment sample that will grow mainly depend on theparticular conditions It is reported that only 1 of microbescan be cultured in the laboratory using traditional culturetechniques [1]

To study the microbial community microbiologists haveadopted culture-independent techniques These techniquesemploy molecular biology based methods in which soil

extracted nucleic acid is subjected to PCR amplification[2] These methods provide a unique insight into richnesscomposition and structure of microbial community thatis species richness and species evenness The results thusrely not only on DNA extraction procedures but also onthe factors affecting PCR amplification Moreover theseculture-independent methods should address the problemslike incomplete rupturing of cells and presence of soil organicsubstances namely fulvic and humic acid the presence ofwhich inhibit the activity of DNA polymerase and interferewith the hybridization protocols [3] Fractions like humicacids are usually the complex mixtures of related compounds(DNA) demonstrating a broad range spectrum of solubilityand charge characteristics Various physical and chemicaltreatments have been evaluated for cell rupture whichinclude shaking the sample in lysis buffers containing highconcentration of sand detergents or glass beads inclusion oflysozyme [4] Furthermore purification of silica and otherbiogel columns has been reported to minimize the humicacid contamination These procedures however make DNAisolation process expensive involving a large number of steps

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2014 Article ID 518960 6 pageshttpdxdoiorg1011552014518960

2 Molecular Biology International

which makes these procedures lengthy time-consumingand tedious Therefore an improved method is required forsoil DNA extraction that would allow efficient rupturing ofmicrobial cells and simultaneously decrease the contamina-tion of organic materials (humic acid) in an easy and cost-effective manner

The analysis of microbial diversity in the soil DNAextracts is then based on ARDRA-amplified ribosomalDNA restriction analysis [5] DGGE-denaturing gradient gelelectrophoresis and ldquoTrdquo-RFLP-Transfer restriction fragmentlength polymorphism [6] However random amplificationof polymorphic DNA (RAPD) technique is preferred asthe above described techniques may not amplify fragmentsfrom all community members with equal effectiveness Suchapproach thus offers significant advantage over just 1 ofthe microbial community accessible with standard culture-based techniques An additional advantage is that only smallamount of soil sample is required for analyzing microbialdiversity in a short span of time

In the present study four DNA extraction methods anda commercial Soil Master DNA extraction kit were used toextract DNA directly from soil and the effectiveness of thesemethods was estimated by RAPD analysis

2 Material and Methods

21 DNA Extraction Methods Five DNA extraction methodswere evaluated in this study with respect to the quality andpurity of extractedDNAusing single type of rhizospheric soilThree modified mannitol-based methods [7] polyethyleneglycol (PEGNaCl) method [8] and a soil DNA extractionkit were compared for obtaining a high recovery and DNAwith good yield and purity Isolated soil samples were isolatedand immediately placed on dry ice mixed and then stored atminus20∘C prior to DNA extraction

211 DNA Extraction Using Polyethylene Glycol (PEG)NaClMethod One gram of soil sample was mixed with 10mLof DNA extraction buffer (120mM Na

2HPO4(pH 74)

5 SDS (wv) and 002 g PVPP) in centrifuge tubes andincubated for 1 h at 65∘C with occasional stirring Thesupernatant was collected after centrifugation at 8000 rpmfor 10min at 4∘C and mixed with half volume of PEG and 1volume of NaCl and incubated at 4∘C for overnight Further 1volume of chloroform isoamyl alcohol (24 1) was added andcentrifuged at 12000 rpm for 10min at 4∘C The supernatantobtained was precipitated by addition of 110th volume of 3Msodium acetate (pH 52) and 2 volumes of ethanol Finally thepellet was recovered by centrifugation at 12000 rpm at 4∘Cand dissolved in 25 120583L TE buffer (Tris-HCl 10mM (pH 78)EDTA 1mM (pH 8))

212 DNA Extraction Using Soil DNA Extraction Kit (SoilMaster DNA Extraction Kit) DNA was extracted from soilsample (1 gm) according to the specifications of the supplier(EPICENTRE Madison WI USA) The method involveddirect cell lysis with prewarmed 5mL solution A at 65∘C andvortexing for 10min This mixture was incubated for 15min

at 65∘C The supernatants were collected after centrifugationat 8000 rpm at 4∘C for 10min and mixed with equal volumeof solution B that led to precipitation The steps wererepeated two times when the color of supernatant changedto yellow Finally the pellet was recovered by centrifugation at12000 rpm for 10min at 4∘C and dissolved in 25120583L TE buffer

213 Modified Mannitol-Based Methods One gram of soilsample was ground using liquid nitrogen This was followedby addition of 5mL of 120mM phosphate buffer saline (pH74) and shaking at 150 rpm for 10min at 4∘C The soilsuspensionwas centrifuged at 7000 rpm for 10minThepelletwas rewashed with PBS buffer and suspended in 10mL ofDNA extraction buffer containing 1MTris-HCl (pH 80) 5MNaCl 05M EDTA (pH 80) 10 CTAB 10 SDS and 02Mmannitol The suspension was incubated for 1 h at 65∘C withoccasional stirring of 150 rpm and subjected to three differenttreatments as indicated

(i) DNA Extraction by Mannitol-Phosphate Buffer Saline-Polyethylene GlycolSodium Chloride (Mannitol-PBS-PEGNaCl) Method The soil suspension described abovewas centrifuged at 8000 rpm for 10min at 4∘C and thesupernatant obtained was mixed with half volume ofpolyethylene glycol-8000 (50) (PEG) and 1 volume of NaCland allowed to incubate at 4∘C for overnight The pelletwas recovered by centrifugation at 12000 rpm at 4∘C for10min and dissolved in 3mL of TE buffer The DNA samplewas then purified by phenol chloroform extraction FinallyDNA was precipitated by addition of 110th volume of 3Msodium acetate (pH 52) and 2 volumes of ethanol The pelletwas recovered by centrifugation at 12000 rpm for 10min at4∘C and dissolved in 25120583L TE buffer (Tris-HCl 10mM (pH78) EDTA 1mM (pH 8))

(ii) DNA Extraction by Mannitol-Phosphate Buffer Saline-PhenolChloroformIsoamyl Alcohol (Mannitol-PBS-PCI)Method After centrifuging the soil suspension as previouslydescribed the supernatant thus obtained was extracted withan equal volume of PCI by centrifugation at 12000 rpm for10min at at 4∘C Aqueous fraction was mixed with 110thvolume of 3M sodium acetate (pH 52) and 2 volumes of 70chilled ethanol at 4∘C Finally the pellet was recovered bycentrifugation at 12000 rpm for 10min at 65∘C and dissolvedin 25 120583L TE buffer (Tris-HCl 10mM (pH 78) EDTA 1mM(pH 8))

(iii) DNA Extraction by Mannitol-Phosphate Buffer Saline-Cetrimide (Mannitol-PBS-CTAB) Method After centrifuga-tion of soil suspension 50120583L of 5M NaCl and 50 120583L of 10CTAB (cetrimide prepared in 07M NaCl) were added to thesupernatant and incubated at 4∘C for 15min This was fol-lowed by addition of equal volume of PCI and centrifugationat 12000 rpm at 4∘C overnight Aqueous layer was allowed toprecipitate overnight at 4∘Cwith 110th volumeof 3M sodiumacetate (pH 52) and 2 volumes of ethanol Finally the pelletwas recovered by centrifugation at 12000 rpm for 10min at65∘C and dissolved in 25120583L TE buffer (Tris-HCl 10mM (pH78) EDTA 1mM (pH 8))

Molecular Biology International 3

22 DNA Yield and Purity The DNA concentration of thesoil sample was measured by examining the absorbance ofthe sample at 260 nm and the amount of DNAwas calculated(10 A

260unit = 50120583gmL of DNA) [8] The purity of the

extracted DNA was determined by taking absorbance at 230260 and 280 nm A pure sample of DNA has the A

260A280

ratio as 18 and the A260

A230

ratio as 20 whereas DNApreparation that is contaminated with protein will have anA260

A280

ratio lower than 18 [9]

23 PCR Amplification of Isolated Soil DNA Using 16S rRNAPrimers for Bacterial Identification Soil DNA was amplifiedby PCR using a PCR BIORAD Thermal Cycler (UnitedKingdom) Each 25 120583L PCR mixture contained 1 120583L (1 10dilution) community DNA (10 ngndash20 ng) 25120583L PCR buffer(1X) 1 120583L of each deoxyribonucleoside triphosphate (dNTP)(100mM) 1 120583L of forward and reverse primers (05 120583M)and 05 120583L Taq polymerase (3U) (Fermentas) The 16S rRNAregions were amplified by using 16S rRNA primers namely(FP1) 51015840-TGGGGAGCAAACAGGATTAG-31015840 and (RP1) 51015840-TAAGGTTCTTCGCTTGCTT-31015840 The amplification cycleconsisted of an initial denaturation step of 30min at 94∘Cfollowed by 35 cycles of 1min at 94∘C (denaturation) 1min at55∘C (annealing) and 2min at 72∘C (extension) with a finalextension step for 5min at 72∘C For visualizing PCR prod-ucts 5 120583L of the amplified product was electrophoresed on 1agarose gel in 1X TAE buffer stained with ethidium bromide(EtBr 05 120583gmL) and analyzed by gel documentation system(BIORAD) Lambda DNA EcoRIHind-III double digest wasused as a molecular size marker

24 PCR Amplification of Soil DNA Extract Using 18S rRNAPrimers for Fungal Identification Soil DNA was submittedfor PCR amplification by using PCR BIORAD ThermalCycler (United Kingdom) A region from 18S rRNA gene wasamplified using internal transcribed spacer (ITS) primersnamely ITS 5 (51015840-GGAAGTAAAAGTCGTAACAAGG-31015840)and ITS 4 (51015840-TCCTCCGCTTATTGATATGC-31015840) Each25 120583L reaction mixture contained 2 120583L soil DNA (10 ngndash20 ng) 25 120583L PCR buffer (1X) 05 120583L deoxyribonucleosidetriphosphate (dNTP mix) (100mM) 05120583L of forward andreverse primers (02 120583M) 2 120583L ofMgCl

2(25mM) and 05 120583L

Taq polymerase (3U) (Fermentas) The amplification cycleconsisted of an initial denaturation step of 5min at 94∘Cfollowed by 35 cycles of 1min at 94∘C (denaturation) 1min at59∘C (annealing) and 2min at 72∘C (extension) with a finalextension step for 10min at 72∘C

25 Random Amplification of Polymorphic DNA (RAPD) Totest the efficiency of soil DNA extraction methods RAPDwas performed on community DNA Four decameric RAPDprimers namely OPA 3 OPA 13 OPA 15 and OPA 20(Operon Technologies) were investigated (Table 1) Randomprimers are short DNA fragments of arbitrary nucleotidesequence that can differentiate between genetically distinctindividuals The RAPD analysis was carried out throughPCR amplification of total DNA Amplification reactionswere performed in a total volume of 25 120583L containing 25 120583L

Table 1 Random primers used for RAPD analysis and theirannealing temperature

Random primer Primer sequences Annealing temperatureOPA 3 51015840-AGTCAGCCAC 32∘COPA 13 51015840-CAGCACCCAC 32∘COPA 15 51015840-TTCCGAACCC 32∘COPA 20 51015840-GTTGCGATCC 32∘C

PCR-buffer (10X) 25 120583L dNTP mix (2mM) 1120583L decamericprimers (20 pmole) 2 120583L template (soil) DNA (100 ng)and 05 120583L Taq DNA polymerase (3U) The final volumewas made up to 25 120583L using sterile distilled water Theamplification reaction was performed for 45 cycles and eachcycle comprised of 3min at 94∘C (denaturation) 1min at32∘C (annealing) and 2min at 72∘C (extension) with a finalextension at 72∘C for 10min

26 Analysis of PCR Products For visualizing PCR products5 120583L of the amplified product was electrophoresed on 1agarose gel in 1X TAE buffer stained with ethidium bromide(05 120583gmL) and analyzed by Gel Documentation system(BIORAD) Lambda DNA EcoRIHind-III double digest wasused as a molecular size marker

3 Results and Discussion

For soil microbial analysis it is essential to design protocolswhich yield high quality soil DNA of appropriate yield andpurity for PCR amplifications Besides the selected methodsfor soil DNA extraction should be cost-effective and time-saving Effectiveness of soil DNA extraction procedures maybe influenced by various parameters such as incomplete celllysis DNA sorption to soil surfaces extraction of humiccontaminants and DNA degradation Thus extraction ofhigh molecular weight DNA proper lysis of microbes andinhibitor-free DNA are the major requirements for anyprotocol used for metagenomic study [10]

For cell lysis to be effective mechanical treatment shouldbe followed rather than chemical ones [11] According toFrostegard et al [4] proper grinding of sample ruptures thecell wall thereby releasing the cellular DNA from the innercompartment

Soil DNA extraction procedures should therefore be freefrom PCR inhibitors or their concentration must be lowenough so that they do not interfere with the enzymatic reac-tions Usually organic matter is the major source of inhibitorsthatmay be coextractedwith themicrobial DNApresent within the soil Majorly humic acids create considerable problemlike interference in activity of DNA polymerase used for PCRreactions [12] As humic acid contains the same charge andsize characteristics like DNA it exhibits absorbance at both230 and at 260 nm and hence interferes in quantization ofDNA This characteristic can be used to find out the level ofcontamination of humic acid in an isolated DNA sample

The present study involved comparison of five methodsfor isolation of soil DNA Three methods having mannitol


Table 2 Comparison of amount purity of DNA and humic acidcontamination extracted from various isolation protocols

DNA extraction protocolAmount of

DNA(120583gmL)

A260230

A260280

DNA extraction using PEGNaClmethod [8] 073 112 126

DNA extraction usingcommercial soil DNA extractionkit (Soil Master DNA extractionkit EPICENTRE Madison WIUSA)

079 121 132

DNA extraction bymannitol-PBS-PEGNaClmethod

220 184 181

DNA extraction bymannitol-PBS-PCI method 236 193 184

DNA extraction bymannitol-PBS-CTAB method 267 207 185

2 3 4 51

Figure 1 Visualization of soil DNA extracted by various methodsLane 1 PEGNaCl method without liquid nitrogen lane 2 SoilMaster DNA extraction kit lane 3 mannitol-PBS-CTAB methodlane 4mannitol-PBS-PCImethod lane 5mannitol-PBS-PEGNaClmethod

in their extraction buffer yielded an amount of DNA thatwas significantly higher than that obtained with the SoilMaster DNA extraction kit and PEGNaCl method (Table 2)Moreover the purity of DNA isolated by modified mannitol-based methods was significantly higher as compared to othermethods (Table 2)The addition ofmannitol in the extractionbuffer has already been reported to enhance the efficiencyof soil DNA extraction [7] Further modification of thesemethods by inclusion of 120mM phosphate buffered salinein the initial steps led to reduction in the level of organiccontaminants such as humic acid at initial stages (Table 2)Soil Master DNA extraction kit and PEGNaCl method withliquid nitrogen method consistently extracted DNA withhigher A

260230and A

260280ratios thereby indicating that

theDNAwas contaminatedwith humic acid-like compoundsand proteins respectively A

260230ratio of more than 20

was obtained with all the three mannitol-based methodswhich was indicative of the fact that humic acid material veryeffectively reduced by thesemethods as compared to the othertwo methods

The three modified mannitol-based methods led to therecovery of high molecular weight soil DNA (Figure 1)The recovery of high molecular weight DNA fraction is

2 3 4 51M

21226

5148

2353

1375

947

831

564

sim12 kbp

Figure 2 Visualization of PCR amplification products of soil DNAisolated by five different methods using 16S rRNA by differentmethods LaneM 120582DNA EcoRIHind III double digestmarker lane1 mannitol-PBS-PCI method lane 2 Soil Master DNA extractionkit lane 3 mannitol-PBS-CTABmethod lane 4 PEGNaCl methodwithout liquid nitrogen lane 5 mannitol-PBS-PEGNaCl method

2 3 4 51M

21226

514823531375947831564 sim720bp

Figure 3 Visualization of PCR amplification products of soil DNAisolated by five different methods using 18S rRNA by differentmethods LaneM 120582DNA EcoRIHind III double digestmarker lane1 mannitol-PBS-CTAB method lane 2 PEGNaCl method withoutliquid nitrogen lane 3 mannitol-PBS-PEGNaCl method lane 4mannitol-PBS-PCI method lane 5 Soil Master DNA extraction kit

desirable for PCR assays used for microbial diversity analysisbecause the degradedDNAupholds the formation of chimeraproducts Moreover these three modified mannitol-basedmethods were found to bemore suited for PCR amplificationNuclear rRNA genes have been useful in the molecular studyof bacterial and fungal diversity [13]

High quality PCR amplicons with higher yields wereobserved in case of these three methods using 16S rRNA-specific and ITS-specific primers (ITS1ITS4) for bacterialand fungal analysis respectively In each case amplified prod-ucts corresponded to expected sizes according to primersused A single amplification product of sim12 Kbp for bacteria(Figure 2) and sim720 bp for fungi (Figure 3) was obtainedIn case of the other two methods namely PEGNaCl withliquid nitrogen method and Soil Master DNA extraction kitan acceptable level of DNA was amplified for bacterial com-munity (Figure 2 lanes 1 and 4) but these two methods werenot suitable for PCR amplification for fungal study (Figure 3lanes 2 and 5) Usually an enzyme DNA polymerase used inamplification processes requires contamination-free sites forproper functioning Furthermore better PCR amplificationof soil DNA isolated by the three modified mannitol-based


2 3 4 51M

(a)

2 3 4 51M

(b)

Figure 4 RAPD analysis of soil DNA samples isolated by five methods using random decameric primers (a) OPA 3 and (b) OPA 13 LaneM120582 DNA EcoRIHind III double digest marker lane 1 Soil Master DNA extraction kit lane 2 mannitol-PBS-CTAB method lane 3 mannitol-PBS-PEGNaCl method lane 4 mannitol-PBS-PCI method lane 5 PEGNaCl method without liquid nitrogen

2 3 4 51 M

(a)

2 3 4 51 M

(b)

Figure 5 RAPD analysis of soil DNA samples isolated by five methods using random decameric primers (a) OPA 15 and (b) OPA 20 LaneM 120582 DNA EcoRIHind III double digest marker lane 1 PEGNaCl method without liquid nitrogen lane 2 mannitol-PBS-CTAB methodlane 3 Soil Master DNA extraction kit lane 4 mannitol-PBS-PCI method lane 5 mannitol-PBS-PEGNaCl method

methods demonstrated better DNA yield and quality ascompared to the other two methods The study suggestedthat all three mannitol-based methods (PCI PEGNaCl andCTAB) gave very good yield of DNA which was suitable forthe amplification study in comparison with the other twomethods which might be due to DNA-adhering substanceslike humicfulvic acid having the same charge characteristicsas those of DNA that were coprecipitated in the mannitol-devoid methods Humic acid impurities may affect DNAhybridization efficiency too The method in which man-nitol with liquid nitrogen was used showed high-qualitychemical lysis as compared to other methods It also provesthat an addition of PBS bufffer and mannitol may playan important role in proper chemical lysis of the cells ascompared to other chemicals like CTAB SDS EDTA and soforth

Thus thesemethods proved to be a low-cost and practicalalternative to accessing metagenomic content by addition ofphosphate buffer (PBS) and mannitol within the soil sample

Varying patterns of RAPD bands were found when soilcommunity DNA samples were amplified using random

primersThis indicated that the reported soil DNA extractionmethods were quite feasible and reproducible for microbialdiversity analysis (Figures 4 and 5) It was observed that thesoilDNA isolated by protocols havingmannitolwas amplifiedeasier than the methods using PEGNaCl and soil DNAextraction kit

Thus the procedure presented here proves to be an inex-pensive procedure which not only prevents the loss of DNAbut also reduces the risk of contamination by laboratoryDNAsource The protocols involved the usage of mannitol withinthe lysis buffer to isolate DNA from bacterial and fungalmycelia The methods used in the present study exhibitedsufficient quality and integrity to amplify the genetic regionswhich provided a complete information and understandingof microbial biota An inclusion of mannitol and sodiumchloride promoted cell disruption and extracted humic acidand other organic contaminants the presence of whichwouldhave otherwise inhibited PCR reaction Hereby it is demon-strated that a molecular approach using culture independentstudy can be used to complement more traditional methodsused for the survey of microbial communities and provides


an expanding toolbox which helps the soil ecologists andtaxonomists to explore microbial communities which arestill unidentified The modified protocols can also contributeto in situ study of bacterial and fungal ecological processes

4 Conclusion

In the present study efficient soil DNA extraction procedureshave been reported which are simple and efficient anddo not require elaborate instrumentation and yield goodquality DNA suitable for the study of bacterial and fungalgenes It has been demonstrated that an additional step ofusing phosphate buffer saline with inclusion of mannitol wasuseful to achieve these objectives The PCR amplificationprocedures involve several enzymatic reactions where theenzyme DNA polymerase requires sites which should becontamination-free It is suggested that the initial washingwith PBS buffer led to removal of unwanted impuritiessuch as humic acid present in the soil Mannitol havinghigh salt nature led the recovery of high molecular weightDNA It probably interacted with cell wall resulting in celldisruption and extraction of humic acid near the beginningof the isolation procedure Thus these modified mannitol-based protocols help not only in improving the yield andquality of extracted soil DNA but also in exploitation oflarge-scale preparations which provide greater possibility fordetecting genes present in low abundance within the soilenvironment When combined with the methods developedfor normalization of total metagenomicDNA thesemodifiedprotocols may offer an easy method for monitoring thepopulation dynamics of the totalmicrobial population in soilsover time

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The financial support in the form of research grant fromUP State Biodiversity Board India is highly acknowledgedThe grant of UGC-Maulana Azad National Fellowship isgratefully acknowledged

References

[1] J R Marchesi T Sato A J Weightman et al ldquoDesignand evaluation of useful bacterium-specific PCR primers thatamplify genes coding for bacterial 16S rRNArdquo Applied andEnvironmental Microbiology vol 64 no 6 pp 795ndash799 1998

[2] C Roh F Villatte B-G Kim and R D Schmid ldquoCom-parative study of methods for extraction and purification ofenvironmental DNA from soil and sludge samplesrdquo AppliedBiochemistry and Biotechnology vol 134 no 2 pp 97ndash112 2006

[3] H Luo H Qi K Xue andH Zhang ldquoA preliminary applicationof PCR-DGGE to study microbial diversity in soilrdquo ActaEcologica Sinica vol 23 pp 1570ndash1575 2003

[4] A Frostegard S Courtois V Ramisse et al ldquoQuantificationof bias related to the extraction of DNA directly from soilsrdquoApplied and Environmental Microbiology vol 65 no 12 pp5409ndash5420 1999

[5] S Weidner W Arnold and A Puhler ldquoDiversity of unculturedmicroorganisms associated with the seagrass Halophila stipu-lacea estimated by restriction fragment length polymorphismanalysis of PCR-amplified 16S rRNA genesrdquo Applied and Envi-ronmental Microbiology vol 62 no 3 pp 766ndash771 1996

[6] A H Goldstein and S T Liu ldquoMolecular cloning and regu-lation of a mineral phosphate solubilizing gene from Erwiniaherbicolardquo BioTechnology vol 5 no 1 pp 72ndash74 1987

[7] F Fatima I Chaudhary J Ali S Rastogi and N PathakldquoMicrobial DNA extraction from soil by different methods andits PCR amplificationrdquo Biochemical and Cellular Archives vol11 no 1 pp 85ndash90 2011

[8] C Yeates M R Gillings A D Davison N Altavilla and D AVeal ldquoMethods formicrobial DNA extraction from soil for PCRamplificationrdquo Biological Procedures Online vol 1 no 1 pp 40ndash47 1998

[9] J Sambrook and D W Russell Molecular CloningmdashA Labora-toryManual Cold SpringHarbor Laboratory Press Cold SpringHarbor NY USA 2001

[10] H J Gu Y Li and W Zhao ldquoComparison of methods of DNAextraction from paddy soilrdquo Journal of Jiangsu University vol15 pp 300ndash305 2005

[11] M I More J B Herrick M C Silva W C Ghiorse and E LMadsen ldquoQuantitative cell lysis of indigenous microorganismsand rapid extraction ofmicrobial DNA from sedimentrdquoAppliedand Environmental Microbiology vol 60 no 5 pp 1572ndash15801994

[12] S Courtois A Frostegard P Goransson G Depret P Jeanninand P Simonet ldquoQuantification of bacterial subgroups in soilcomparison of DNA extracted directly from soil or fromcells previously released by density gradient centrifugationrdquoEnvironmental Microbiology vol 3 no 7 pp 431ndash439 2001

[13] T R Horton and T D Bruns ldquoThe molecular revolution inectomycorrhizal ecology peeking into the black-boxrdquo Molecu-lar Ecology vol 10 no 8 pp 1855ndash1871 2001

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which makes these procedures lengthy time-consumingand tedious Therefore an improved method is required forsoil DNA extraction that would allow efficient rupturing ofmicrobial cells and simultaneously decrease the contamina-tion of organic materials (humic acid) in an easy and cost-effective manner

The analysis of microbial diversity in the soil DNAextracts is then based on ARDRA-amplified ribosomalDNA restriction analysis [5] DGGE-denaturing gradient gelelectrophoresis and ldquoTrdquo-RFLP-Transfer restriction fragmentlength polymorphism [6] However random amplificationof polymorphic DNA (RAPD) technique is preferred asthe above described techniques may not amplify fragmentsfrom all community members with equal effectiveness Suchapproach thus offers significant advantage over just 1 ofthe microbial community accessible with standard culture-based techniques An additional advantage is that only smallamount of soil sample is required for analyzing microbialdiversity in a short span of time

In the present study four DNA extraction methods anda commercial Soil Master DNA extraction kit were used toextract DNA directly from soil and the effectiveness of thesemethods was estimated by RAPD analysis

2 Material and Methods

21 DNA Extraction Methods Five DNA extraction methodswere evaluated in this study with respect to the quality andpurity of extractedDNAusing single type of rhizospheric soilThree modified mannitol-based methods [7] polyethyleneglycol (PEGNaCl) method [8] and a soil DNA extractionkit were compared for obtaining a high recovery and DNAwith good yield and purity Isolated soil samples were isolatedand immediately placed on dry ice mixed and then stored atminus20∘C prior to DNA extraction

211 DNA Extraction Using Polyethylene Glycol (PEG)NaClMethod One gram of soil sample was mixed with 10mLof DNA extraction buffer (120mM Na

2HPO4(pH 74)

5 SDS (wv) and 002 g PVPP) in centrifuge tubes andincubated for 1 h at 65∘C with occasional stirring Thesupernatant was collected after centrifugation at 8000 rpmfor 10min at 4∘C and mixed with half volume of PEG and 1volume of NaCl and incubated at 4∘C for overnight Further 1volume of chloroform isoamyl alcohol (24 1) was added andcentrifuged at 12000 rpm for 10min at 4∘C The supernatantobtained was precipitated by addition of 110th volume of 3Msodium acetate (pH 52) and 2 volumes of ethanol Finally thepellet was recovered by centrifugation at 12000 rpm at 4∘Cand dissolved in 25 120583L TE buffer (Tris-HCl 10mM (pH 78)EDTA 1mM (pH 8))

212 DNA Extraction Using Soil DNA Extraction Kit (SoilMaster DNA Extraction Kit) DNA was extracted from soilsample (1 gm) according to the specifications of the supplier(EPICENTRE Madison WI USA) The method involveddirect cell lysis with prewarmed 5mL solution A at 65∘C andvortexing for 10min This mixture was incubated for 15min

at 65∘C The supernatants were collected after centrifugationat 8000 rpm at 4∘C for 10min and mixed with equal volumeof solution B that led to precipitation The steps wererepeated two times when the color of supernatant changedto yellow Finally the pellet was recovered by centrifugation at12000 rpm for 10min at 4∘C and dissolved in 25120583L TE buffer

213 Modified Mannitol-Based Methods One gram of soilsample was ground using liquid nitrogen This was followedby addition of 5mL of 120mM phosphate buffer saline (pH74) and shaking at 150 rpm for 10min at 4∘C The soilsuspensionwas centrifuged at 7000 rpm for 10minThepelletwas rewashed with PBS buffer and suspended in 10mL ofDNA extraction buffer containing 1MTris-HCl (pH 80) 5MNaCl 05M EDTA (pH 80) 10 CTAB 10 SDS and 02Mmannitol The suspension was incubated for 1 h at 65∘C withoccasional stirring of 150 rpm and subjected to three differenttreatments as indicated

(i) DNA Extraction by Mannitol-Phosphate Buffer Saline-Polyethylene GlycolSodium Chloride (Mannitol-PBS-PEGNaCl) Method The soil suspension described abovewas centrifuged at 8000 rpm for 10min at 4∘C and thesupernatant obtained was mixed with half volume ofpolyethylene glycol-8000 (50) (PEG) and 1 volume of NaCland allowed to incubate at 4∘C for overnight The pelletwas recovered by centrifugation at 12000 rpm at 4∘C for10min and dissolved in 3mL of TE buffer The DNA samplewas then purified by phenol chloroform extraction FinallyDNA was precipitated by addition of 110th volume of 3Msodium acetate (pH 52) and 2 volumes of ethanol The pelletwas recovered by centrifugation at 12000 rpm for 10min at4∘C and dissolved in 25120583L TE buffer (Tris-HCl 10mM (pH78) EDTA 1mM (pH 8))

(ii) DNA Extraction by Mannitol-Phosphate Buffer Saline-PhenolChloroformIsoamyl Alcohol (Mannitol-PBS-PCI)Method After centrifuging the soil suspension as previouslydescribed the supernatant thus obtained was extracted withan equal volume of PCI by centrifugation at 12000 rpm for10min at at 4∘C Aqueous fraction was mixed with 110thvolume of 3M sodium acetate (pH 52) and 2 volumes of 70chilled ethanol at 4∘C Finally the pellet was recovered bycentrifugation at 12000 rpm for 10min at 65∘C and dissolvedin 25 120583L TE buffer (Tris-HCl 10mM (pH 78) EDTA 1mM(pH 8))

(iii) DNA Extraction by Mannitol-Phosphate Buffer Saline-Cetrimide (Mannitol-PBS-CTAB) Method After centrifuga-tion of soil suspension 50120583L of 5M NaCl and 50 120583L of 10CTAB (cetrimide prepared in 07M NaCl) were added to thesupernatant and incubated at 4∘C for 15min This was fol-lowed by addition of equal volume of PCI and centrifugationat 12000 rpm at 4∘C overnight Aqueous layer was allowed toprecipitate overnight at 4∘Cwith 110th volumeof 3M sodiumacetate (pH 52) and 2 volumes of ethanol Finally the pelletwas recovered by centrifugation at 12000 rpm for 10min at65∘C and dissolved in 25120583L TE buffer (Tris-HCl 10mM (pH78) EDTA 1mM (pH 8))





260A280


A230


A280




2(25mM) and 05 120583L















DNA(120583gmL)

A260230

A260280



079 121 132


220 184 181



2 3 4 51



260230and A






2 3 4 51M

21226

5148

2353

1375

947

831

564

sim12 kbp


2 3 4 51M

21226

514823531375947831564 sim720bp





2 3 4 51M

(a)

2 3 4 51M

(b)


2 3 4 51 M

(a)

2 3 4 51 M

(b)









4 Conclusion




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





260A280


A230


A280




2(25mM) and 05 120583L















DNA(120583gmL)

A260230

A260280



079 121 132


220 184 181



2 3 4 51



260230and A






2 3 4 51M

21226

5148

2353

1375

947

831

564

sim12 kbp


2 3 4 51M

21226

514823531375947831564 sim720bp





2 3 4 51M

(a)

2 3 4 51M

(b)


2 3 4 51 M

(a)

2 3 4 51 M

(b)









4 Conclusion




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




DNA(120583gmL)

A260230

A260280



079 121 132


220 184 181



2 3 4 51



260230and A






2 3 4 51M

21226

5148

2353

1375

947

831

564

sim12 kbp


2 3 4 51M

21226

514823531375947831564 sim720bp





2 3 4 51M

(a)

2 3 4 51M

(b)


2 3 4 51 M

(a)

2 3 4 51 M

(b)









4 Conclusion




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2 3 4 51M

(a)

2 3 4 51M

(b)


2 3 4 51 M

(a)

2 3 4 51 M

(b)









4 Conclusion




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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