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DNA extraction method affects microbial community profiles from soils and sediment

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Abstract

To evaluate whether different deoxyribonucleic acid (DNA) extraction procedures can affect estimates of bacterial community composition, based on the 16S ribosomal ribonucleic acid gene denaturing gradient gel electrophoresis (DGGE) profiles, we compared four in situ lysis procedures using three soils and one marine sediment. Analysis of DGGE profiles, generated by polymerase chain reaction of purified DNA extracts, demonstrated that the choice of DNA extraction method significantly influenced the bacterial community profiles generated. This was reflected both in the number of bands or ribotypes detected from each sample and in subsequent principle coordinate analysis and unweighted-pair group method using arithmetic average analyses. The methods also differed significantly in their robustness, i.e. reproducibility across multiple analyses. Two methods, both based on bead beating, were demonstrated to be suitable for comparative studies of a range of soil and sediment types.

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References

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in-situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blackwood CB, Paul EA (2003) Eubacterial community structure and population size within the soil light fraction, rhizosphere, and heavy fraction of several agricultural systems. Soil Biol Biochem 35:1245–1255

    Article  CAS  Google Scholar 

  • Bürgmann H, Pesaro M, Widmer F, Zeyer J (2001) A strategy for optimizing quality and quantity of DNA extracted from soil. J Microbiol Methods 45:7–20

    Article  PubMed  Google Scholar 

  • de Lipthay JR, Enzinger C, Johnsen K, Aamand J, Sørensen SJ (2004) Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis. Soil Biol Biochem 36:1607–1614

    Article  CAS  Google Scholar 

  • DeLong EF, Franks DG, Allredge ADL (1993) Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Limnol Oceanogr 38:924–934

    Article  Google Scholar 

  • Demanéche S, Jocteur-Monrozier L, Quiquampoix H, Simonet P (2001) Evaluation of biological and physical protection against nuclease degradation of clay-bound plasmid DNA. Appl Environ Microbiol 67:293–299

    Article  PubMed  PubMed Central  Google Scholar 

  • Dunbar J, Ticknor LO, Kuske CR (2001) Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. J Environ Microbiol 67:190–197

    Article  CAS  Google Scholar 

  • Eardly DF, Carton MW, Gallagher JM, Patching JW (2001) Bacterial abundance and activity in deep-sea marine sediments from the Eastern North Atlantic. Prog Oceanogr 50:245–259

    Article  Google Scholar 

  • Ferris MJ, Ward M (1997) Seasonal distributions of dominant 16S rRNA-defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis. Appl Environ Microbiol 63:1375–1381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fromin N, Hamelin J, Tarnawski S, Roesti P, Jourdain-Miserez K, Forestier N, Teyssier-Cuvelle S, Gillet F, Aragzo M, Rossi P (2002) Statistical analysis of denaturing gradient elctrophoresis fingerprinting patterns. Environ Microbiol 4(11):634–643

    Article  CAS  PubMed  Google Scholar 

  • Frostegård Å, Courtois S, Ramisse V, Clerc S, Bernillon D, Le Gall F, Jeannin P, Nesme X, Simonet P (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 65:5409–5420

    Article  PubMed  PubMed Central  Google Scholar 

  • Gabor EM, de Vries EJ, Janssen DB (2003) Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiol Ecol 44:153–163

    Article  CAS  PubMed  Google Scholar 

  • Grayston SJ, Vaughan D, Jones D (1996) Rhizosphere carbon flow in trees, in comparison with annual plans: the importance of root exudation and its impact on microbial activity and nutrient availability. J Soil Ecol 5:29–56

    Article  Google Scholar 

  • Grayston SJ, Campbell CD, Bardgett RD (2004) Assessing shifts in microbial community structure across a range of grasslands of differing management intensity using CLPP, PLFA and community DNA techniques. J Soil Ecol 25:63–84

    Article  Google Scholar 

  • Griffiths RI, Whitely AS, O’Donnell AG, Bailey MJ (2000) Rapid method for Co-extraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition. Appl Environ Microbiol 66:5488–5491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Griffiths RI, Whitely AS, O’Donnell AG, Bailey MJ (2002) Influence of depth and sampling time on bacterial community structure in an upland grassland soil. FEMS Microbiol Ecol 43:35–43

    Article  Google Scholar 

  • Hill GT, Mitkowski NA, Aldrich-Wolfe L, Emele LR, Jurkonie DD, Ficke A, Maldonado-Ramirez S, Lynch ST, Nelson EB (2000) Methods for assessing the composition and diversity of soil microbial communities. Appl Soil Ecol 15:25–36

    Article  Google Scholar 

  • Howeler M, Ghiorse WC, Walker LP (2003) A quantitative analysis of DNA extraction and purification from compost. J Microbiol Methods 54:37–45

    Article  CAS  PubMed  Google Scholar 

  • Ibekwe AM, Kennedy AC, Frohne PS, Papiemik SK, Yang CH, Crowley DE (2002) Microbial diversity along a transect of agronomic zones. FEMS Microbiol Ecol 39(3):183–191

    Article  CAS  PubMed  Google Scholar 

  • Kisand V, Wikner J (2003) Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences. J Microbiol Methods 54:183–191

    Article  CAS  PubMed  Google Scholar 

  • Kozdrόz J, van Elsas JD (2000) Application of polymerase chain reaction-denaturing gradient electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting. Biol Fertil Soils 31:372–378

    Article  Google Scholar 

  • Krsek M, Wellington EMH (1999) Comparison of different methods for the isolation and purification of total community DNA from soil. J Microbiol Methods 39:1–16

    Article  CAS  PubMed  Google Scholar 

  • Kuske CR, Banton KL, Adorada DL, Stark PC, Hill KK, Jackson PJ (1998) Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil. Appl Environ Microbiol 64:2463–2472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • LaMontagne MG, Michel Jr FC, Holden PA, Reddy CA (2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods 49:255–264

    Article  CAS  PubMed  Google Scholar 

  • Leff G, Dana JR, McArthur JV, Shimkets LJ (1995) Comparison of methods of DNA extraction from stream sediments. Appl Environ Microbiol 61:1141–1143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lloyd-Jones G, Hunter DWF (2001) Comparison of rapid DNA extraction methods applied to contrasting New Zealand soils. Soil Biol Biochem 33:2053–2059

    Article  CAS  Google Scholar 

  • Lornez MG, Wackernagel W (1987) Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Appl Environ Microbiol 53:2948–2952

    Article  Google Scholar 

  • Luna GM, Dell’Anno A, Danovaro R (2006) DNA extraction procedure: a critical issue for bacterial assessment in marine sediments. Environ Microbiol 8:308–320

    Article  CAS  PubMed  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  • Martin-Laurent F, Philippot L, Hallet S, Chaussod R, Germon JC, Soulas G, Catroux G (2001) DNA extraction from soils: Old bias for new microbial diversity analysis methods. Appl Environ Microbiol 67:2354–2359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Menking DE, Emanuel PA, Valdes JJ, Kracke SK (1999) Rapid cleanup of bacterial DNA from field samples. Resour Conserv Recy 27:179–186

    Article  Google Scholar 

  • Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA purification procedures for soil and sediment samples. Appl Environ Microbiol 65:4715–424

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moré MI, Herrick JB, Silva MC, Ghiorse WC, Madsen EL (1994) Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl Environ Microbiol 60:1572–1580

    Article  PubMed  PubMed Central  Google Scholar 

  • Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S ribosomal-RNA. Appl Environ Microbiol 59:695–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nannipieri P, Ascher J, Ceccherini MT, Landi L, Pietramellara G, Renella G (2003) Microbial diversity and soil functions. Eur J Soil Sci 54:655–670

    Article  Google Scholar 

  • Nielsen MN, Winding A (2002) Microorganisms as indicators of soil health. Technical Report no. 388. National Environmental Research Institute, Denmark

    Google Scholar 

  • Niemi RM, Heiskanen I, Wallenius K, Lindström K (2001) Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia. J Microbiol Methods 45:155–165

    Article  Google Scholar 

  • Nunan N, Wu K, Young IM, Crawford JM, Ritz K (2003) Spatial distribution of bacterial communities and their relationship with the micro-architecture of soil. FEMS Microbiol Ecol 44:203–215

    Article  CAS  PubMed  Google Scholar 

  • O’Donnell AG, Gorres HE (1999) 16S rDNA methods in soil microbiology. Curr Opin Biotechnol 10(3):225–229

    Article  PubMed  Google Scholar 

  • Ogram A (2000) Soil molecular microbial ecology at age 20: methodological challenges for the future. Soil Biol Biochem 32:1499–1504

    Article  CAS  Google Scholar 

  • Olsen GJ, Lane DJ, Giovannoni J, Pace NR, Stahl DA (1986) Microbial ecology and evolution: a ribosomal RNA approach. Ann Rev Microiol 40:337–365

    Article  CAS  Google Scholar 

  • Osborn AM, Moore ERB, Timmis KN (2000) An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ Microbiol 2:39–50

    Article  CAS  PubMed  Google Scholar 

  • Robe P, Nalin R, Capellano C, Vogel TM, Simonet P (2003) Extraction of DNA from soil. Eur J Soil Biol 39:183–190

    Article  CAS  Google Scholar 

  • Rochelle PA, Fry JC, Parkes RJ, Weightman AJ (1992) DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sea sediment communities. FEMS Microbiol Lett 100:59–66

    Article  CAS  PubMed  Google Scholar 

  • Roose-Amsaleg CL, Garnier-Sillam E, Harry M (2001) Extraction and purification of microbial DNA from soil and sediment samples. Appl Soil Ecol 18:47–60

    Article  Google Scholar 

  • Sandaa R-A, Enger Ø, Torsvik V (1998) Rapid method for fluorometric quantification of DNA in soil. Soil Biol Biochem 30:265–268

    Article  CAS  Google Scholar 

  • Schloss PD, Handelsman J (2003) Biotechnological prospects from metagenomics. Curr Opin Biotech 14:303–310

    Article  CAS  PubMed  Google Scholar 

  • Scow KM, Bruns MA, Graham K, Bossio D, Schwartz E (1998) Development of indices of microbial community structure for soil quality assessment. In: Zabel A, Sposito G (eds) Soil quality in the California environment. Kearny Foundation of Soil Science Annual Report of Research Projects 1997–1998, pp 110–123

  • Smit E, Leeflang P, Gommans S, van den Broek J, van Mil S, Wernars K (2001) Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. J Environ Microbiol 67:2284–2291

    Article  CAS  Google Scholar 

  • Steffan RJ, Goksoyr J, Bej AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Terashima K, Matsumoto T, Hasebe K, Fukumasa-Nakai Y (2002) Genetic diversity and strain-typing in cultivated strains of Lentinula edodes (the shii-take mushroom) in Japan by AFLP analysis. Mycol Res 106(1):34–39

    Article  CAS  Google Scholar 

  • Tiedje JM, Asuming-Brempong S, Nüsslein K, Marsh TL, Flynn SJ (1999) Opening the black box of soil microbial diversity. Appl Soil Ecol 13:109–122

    Article  Google Scholar 

  • Torsvik VL (1980) Isolation of bacterial DNA from soil. Soil Biol Biochem 12:15–21

    Article  CAS  Google Scholar 

  • Torsvik V, Øvreås L (2002) Microbial diversity and function in soil: from genes to ecosystems. Curr Opin Microbiol 5:240–245

    Article  CAS  PubMed  Google Scholar 

  • Torsvik V, Sørheim R, Goksøyr J (1996) Total bacterial diversity in soil and sediment communities—a review. J Ind Microbiol 17:170–178

    CAS  Google Scholar 

  • Trevors JT (1992) DNA extraction from soil. Microb Releases 1:3–9

    CAS  Google Scholar 

  • Tsai Y-L, Olson BH (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 57:1070–1074

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weinbauer MG, Beckmann C, Höfle MG (1998) Utility of green fluorescent nucleic acid dyes and aluminium oxide membrane filters for rapid enumeration of soil and sediment bacteria. Appl Environ Microbiol 64:5000–5003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Westergaard K, Müller AK, Christensen S, Bloem J, Sørensen SJ (2001) Effects of tylosin as a disturbance on the soil microbial community. Soil Biol Biochem 33:2061–2071

    Article  CAS  Google Scholar 

  • Zhou J, Burns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zipper H, Buta C, Lämmle K, Brunner H, Bernhagen J, Vitzthum F (2003) Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green l and consequences for the analysis of soils and aquatic sediments. Nucleic Acids Res 31(7):e39

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Dr. Fabio Chinalia for assistance with statistical analysis. This research was financially supported by the Irish Environmental Protection Agency Environmental Research Training and Development Program (2000–2006) project “Towards a National Soil Database (2001-CD/S2-M2).”

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Authors and Affiliations

  1. Microbial Ecology Laboratory, Department of Microbiology and Environmental Change Institute (ECI), National University of Ireland, Galway (NUI, Galway), University Road, Galway, Ireland

    Cora Carrigg & Vincent O’Flaherty

  2. Biomerit Research Centre, Department of Microbiology, University College Cork, Cork, Ireland

    Olivia Rice

  3. DNA Laboratory, National Diagnostics Centre, National University of Ireland, Galway (NUI, Galway), University Road, Galway, Ireland

    Siobhán Kavanagh

  4. Nutrient Group, Max Planck Institute for Marine Microbiology, Celsiusstraße 1, 28359, Bremen, Germany

    Gavin Collins

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  1. Cora Carrigg
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  2. Olivia Rice
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  3. Siobhán Kavanagh
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  4. Gavin Collins
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  5. Vincent O’Flaherty
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Correspondence to Vincent O’Flaherty.

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Fig. 1

PCO analysis of the four extraction methods tested on each of the four soils a Corrib soil, b Silvermines soil, c Inverin peat and d PAP marine sediment. M1–M4 represent methods 1–4. Identical profiles overlap (asterisk represents a profile generated using a separate extract) (JPG 47 kb)

Fig. 2

UPGMA analysis of the four extraction methods tested on each of the four soils a Corrib soil, b Silvermines soil, c Inverin peat and d PAP marine sediment. M1–M4 represent methods 1–4 (asterisk represents a profile generated using a separate extract) (JPG 55 kb)

Fig. 3

PCO analysis of the method reproducibility found with each of the four methods tested on Corrib soil a M1, b M2 , c M3 and d M4. Identical profiles overlap (JPG 25 kb)

Fig. 4

UPGMA analysis of the method reproducibility found with each of the four methods tested on Corrib soil a M1, b M2, c M3 and d M4 (JPG 69 KB)

Fig. 5

PCO analysis of the method reproducibility found with each of the four methods tested on Silvermines soil a M1, b M2, c M3 and d M4. Identical profiles overlap (JPG 25 kb)

Fig. 6

UPGMA analysis of the method reproducibility found with each of the four methods tested on Silvermines soil a M1, b M2, c M3 and d M4 (JPG 71 kb)

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Carrigg, C., Rice, O., Kavanagh, S. et al. DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 77, 955–964 (2007). https://doi.org/10.1007/s00253-007-1219-y

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