Microbial Ecology

, Volume 59, Issue 4, pp 776–786 | Cite as

PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities: A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset

  • Panagiotis Tourlomousis
  • E. Katherine Kemsley
  • Karyn P. Ridgway
  • Michael J. Toscano
  • Thomas J. Humphrey
  • Arjan Narbad


Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.


Terminal Restriction Fragment Length Polymorphism Complex Microbial Community Image Analysis Software Package Densitometric Profile 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by the Department of Environment, Food and Rural Affairs (DEFRA), UK and the Higher Education Funding Council for England (Hefce) under the Veterinary Training and Research Initiative (VTRI).

Supplementary material

248_2009_9613_MOESM1_ESM.docx (1.1 mb)
Figure S1 Raw images of all gels (except one which is shown in Fig. 1) used for the purpose of this study. (DOCX 1078 kb)
248_2009_9613_MOESM2_ESM.docx (45 kb)
Figure S2 Matlab script (m-file) for importing DGGE data output from TL120 in Microsoft Excel format. The raw profiles are interpolated piecewise to common abscissae and collated into a matrix ready for multivariate analysis. The delimiters for each piece are defined in the TL120 alignment process and exported along with the raw profiles in the Excel spreadsheet. (DOCX 44 kb)


  1. 1.
    Alonso-Saez L, Balague V, Sa EL, Sanchez O, Gonzalez JM, Pinhassi J, Massana R, Pernthaler J, Pedros-Alio C, Gasol JM (2007) Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH. FEMS Microbiol Ecol 60:98–112CrossRefPubMedGoogle Scholar
  2. 2.
    Cardinali G, Martini A, Preziosi R, Bistoni F, Baldelli F (2002) Multicenter comparison of three different analytical systems for evaluation of DNA banding patterns from Cryptococcus neoformans. J Clin Microbiol 40:2095–2100CrossRefPubMedGoogle Scholar
  3. 3.
    Duck WM, Steward CD, Banerjee SN, McGowan JE, Tenover FC (2003) Optimization of computer software settings improves accuracy of pulsed-field gel electrophoresis macrorestriction fragment pattern analysis. J Clin Microbiol 41:3035–3042CrossRefPubMedGoogle Scholar
  4. 4.
    Felske A, Osborn AM (2005) DNA fingerprinting of microbial communities. In: Osborn AM, Smith CJ (eds) Molecular microbial ecology. Taylor & Francis, AbingdonGoogle Scholar
  5. 5.
    Ferrari VC, Hollibaugh JT (1999) Distribution of microbial assemblages in the Central Arctic Ocean Basin studied by PCR/DGGE: analysis of a large data set. Hydrobiologia 401:55–68CrossRefGoogle Scholar
  6. 6.
    Ferris MJ, Muyzer G, Ward DM (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62:340–346PubMedGoogle Scholar
  7. 7.
    Forney LJ, Zhou X, Brown CJ (2004) Molecular microbial ecology: land of the one-eyed king. Curr Opin Microbiol 7:210–220CrossRefPubMedGoogle Scholar
  8. 8.
    Fromin N, Hamelin J, Tarnawski S, Roesti D, Jourdain-Miserez K, Forestier N, Teyssier-Cuvelle S, Gillet F, Aragno M, Rossi P (2002) Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environ Microbiol 4:634–643CrossRefPubMedGoogle Scholar
  9. 9.
    Gerner-Smidt P, Graves LM, Hunter S, Swaminathan B (1998) Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages. J Clin Microbiol 36:1318–1323PubMedGoogle Scholar
  10. 10.
    Girvan MS, Bullimore J, Pretty JN, Osborn AM, Ball AS (2003) Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils. Appl Environ Microbiol 69:1800–1809CrossRefPubMedGoogle Scholar
  11. 11.
    Gong J, Yu H, Liu T, Gill JJ, Chambers JR, Wheatcroft R, Sabour PM (2008) Effects of zinc bacitracin, bird age and access to range on bacterial microbiota in the ileum and caeca of broiler chickens. J Appl Microbiol 104:1372–1382CrossRefPubMedGoogle Scholar
  12. 12.
    Green GL, Brostoff J, Hudspith B, Michael M, Mylonaki M, Rayment N, Staines N, Sanderson J, Rampton DS, Bruce KD (2006) Molecular characterization of the bacteria adherent to human colorectal mucosa. J Appl Microbiol 100:460–469CrossRefPubMedGoogle Scholar
  13. 13.
    Konstantinov SR, Zhu WY, Williams BA, Tamminga S, de Vos WM, Akkermans ADL (2003) Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA. FEMS Microbiol Ecol 43:225–235CrossRefPubMedGoogle Scholar
  14. 14.
    Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW (1997) Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 63:1489–1497PubMedGoogle Scholar
  15. 15.
    Kropf S, Heuer H, Gruning M, Smalla K (2004) Significance test for comparing complex microbial community fingerprints using pairwise similarity measures. J Microbiol Methods 57:187–195CrossRefPubMedGoogle Scholar
  16. 16.
    Lanyon CV, Rushton SP, O'Donnell AG, Goodfellow M, Ward AC, Petrie M, Jensen SP, Gosling LM, Penn DJ (2007) Murine scent mark microbial communities are genetically determined. FEMS Microbiol Ecol 59:576–583CrossRefPubMedGoogle Scholar
  17. 17.
    Li M, Wang BH, Zhang MH, Rantalainen M, Wang SY, Zhou HK, Zhang Y, Shen J, Pang XY, Zhang ML, Wei H, Chen Y, Lu HF, Zuo J, Su MM, Qiu YP, Jia W, Xiao CN, Smith LM, Yang SL, Holmes E, Tang HR, Zhao GP, Nicholson JK, Li LJ, Zhao LP (2008) Symbiotic gut microbes modulate human metabolic phenotypes. Proc Natl Acad Sci USA 105:2117–2122CrossRefPubMedGoogle Scholar
  18. 18.
    Liu WT, Marsh TL, Cheng H, Forney LJ (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522PubMedGoogle Scholar
  19. 19.
    Marzotto M, Maffeis C, Paternoster T, Ferrario R, Rizzotti L, Pellegrino M, Dellaglio F, Torriani S (2006) Lactobacillus paracasei A survives gastrointestinal passage and affects the fecal microbiota of healthy infants. Res Microbiol 157:857–866CrossRefPubMedGoogle Scholar
  20. 20.
    McOrist AL, Abell GCJ, Cooke C, Nyland K (2008) Bacterial population dynamics and faecal short-chain fatty acid (SCFA) concentrations in healthy humans. Brit J Nutr 100:138–146CrossRefPubMedGoogle Scholar
  21. 21.
    Michaud S, Menard S, Gaudreau C, Arbeit RD (2001) Comparison of SmaI-defined genotypes of Campylobacter jejuni examined by KpnI: a population-based study. J Med Microbiol 50:1075–1081PubMedGoogle Scholar
  22. 22.
    Moeseneder MM, Arrieta JM, Muyzer G, Winter C, Herndl GJ (1999) Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl Environ Microbiol 65:3518–3525PubMedGoogle Scholar
  23. 23.
    Mouser PJ, Rizzo DM, Roling WFM, Van Breukelen BM (2005) A multivariate statistical approach to spatial representation of groundwater contamination using hydrochemistry and microbial community profiles. Environ Sci Technol 39:7551–7559CrossRefPubMedGoogle Scholar
  24. 24.
    Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  25. 25.
    Nakatsu CH (2007) Soil microbial community analysis using denaturing gradient gel electrophoresis. Soil Sci Soc Am J 71:562–571CrossRefGoogle Scholar
  26. 26.
    Neufeld JD, Mohn WW (2005) Fluorophore-labeled primers improve the sensitivity, versatility, and normalization of denaturing gradient gel electrophoresis. Appl Environ Microbiol 71:4893–4896CrossRefPubMedGoogle Scholar
  27. 27.
    Nielsen DS, Snitkjaer P, van den Berg F (2008) Investigating the fermentation of cocoa by correlating denaturing gradient gel electrophoresis profiles and near infrared spectra. Int J Food Microbiol 125:133–140CrossRefPubMedGoogle Scholar
  28. 28.
    Nocker A, Burr M, Camper AK (2007) Genotypic microbial community profiling: a critical technical review. Microb Ecol 54:276–289CrossRefPubMedGoogle Scholar
  29. 29.
    Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178:5636–5643PubMedGoogle Scholar
  30. 30.
    Nunan N, Daniell TJ, Singh BK, Papert A, McNicol JW, Prosser JI (2005) Links between plant and rhizoplane bacterial communities in grassland soils, characterized using molecular techniques. Appl Environ Microbiol 71:6784–6792CrossRefPubMedGoogle Scholar
  31. 31.
    Paerl HW, Steppe TF (2003) Scaling up: the next challenge in environmental microbiology. Environ Microbiol 5:1025–1038CrossRefPubMedGoogle Scholar
  32. 32.
    Powell SM, Bowman JP, Snape I, Stark JS (2003) Microbial community variation in pristine and polluted nearshore Antarctic sediments. FEMS Microbiol Ecol 45:135–145CrossRefPubMedGoogle Scholar
  33. 33.
    Rehman H, Hellweg P, Taras D, Zentek J (2008) Effects of dietary inulin on the intestinal short chain fatty acids and microbial ecology in broiler chickens as revealed by denaturing gradient gel electrophoresis. Poult Sci 87:783–789CrossRefPubMedGoogle Scholar
  34. 34.
    Rementeria A, Gallego L, Quindos G, Garaizar J (2001) Comparative evaluation of three commercial software packages for analysis of DNA polymorphism patterns. Clin Microbiol Infect 7:331–336CrossRefPubMedGoogle Scholar
  35. 35.
    Roggo Y, Chalus P, Maurer L, Lema-Martinez C, Edmond A, Jent N (2007) A review of near infrared spectroscopy and chemometrics in pharmaceutical technologies. J Pharm Biomed Anal 44:683–700CrossRefPubMedGoogle Scholar
  36. 36.
    Salles JF, van Veen JA, van Elsas JD (2004) Multivariate analyses of Burkholderia species in soil: effect of crop and land use history. Appl Environ Microbiol 70:4012–4020CrossRefPubMedGoogle Scholar
  37. 37.
    Sanchez O, Gasol JM, Massana R, Mas J, Pedros-Alio C (2007) Comparison of different denaturing gradient gel electrophoresis primer sets for the study of marine bacterioplankton communities. Appl Environ Microbiol 73:5962–5967CrossRefPubMedGoogle Scholar
  38. 38.
    Simpson JM, McCracken VJ, Gaskins HR, Mackie RI (2000) Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl Environ Microbiol 66:4705–4714CrossRefPubMedGoogle Scholar
  39. 39.
    Smalla K, Oros-Sichler M, Milling A, Heuer H, Baumgarte S, Becker R, Neuber G, Kropf S, Ulrich A, Tebbe CC (2007) Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: do the different methods provide similar results? J Microbiol Methods 69:470–479CrossRefPubMedGoogle Scholar
  40. 40.
    Szekely A, Sipos R, Berta B, Vajna B, Hajdu C, Marialigeti K (2009) DGGE and T-RFLP analysis of bacterial succession during mushroom compost production and sequence-aided T-RFLP profile of mature compost. Microb Ecol 57:522–533CrossRefPubMedGoogle Scholar
  41. 41.
    Thies JE (2007) Molecular methods for studying soil ecology, 3rd edn. Academic, BurlightonGoogle Scholar
  42. 42.
    Thompson CL, Wang B, Holmes AJ (2008) The immediate environment during postnatal development has long-term impact on gut community structure in pigs. ISME J 2:739–748CrossRefPubMedGoogle Scholar
  43. 43.
    van der Wielen PWJJ, Keuzenkamp DA, Lipman LJA, van Knapen F, Biesterveld S (2002) Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth. Microb Ecol 44:286–293CrossRefPubMedGoogle Scholar
  44. 44.
    von Wintzingerode F, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229CrossRefGoogle Scholar
  45. 45.
    Workman JJ, Mobley PR, Kowalski BR, Bro R (1996) Review of chemometrics applied to spectroscopy: 1985–95.1. Appl Spectrosc Rev 31:73–124CrossRefGoogle Scholar
  46. 46.
    Zoetendal EG, Akkermans ADL, De Vos WM (1998) Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Appl Environ Microbiol 64:3854–3859PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Panagiotis Tourlomousis
    • 1
    • 3
  • E. Katherine Kemsley
    • 2
  • Karyn P. Ridgway
    • 1
  • Michael J. Toscano
    • 3
  • Thomas J. Humphrey
    • 3
  • Arjan Narbad
    • 1
  1. 1.Integrated Biology of GI Tract ProgrammeInstitute of Food ResearchNorwichUK
  2. 2.Bioinformatics and Statistics PartnershipInstitute of Food ResearchNorwichUK
  3. 3.Department of Clinical Veterinary SciencesUniversity of BristolBristolUK

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