Skip to main content
Log in

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

  • Mini-Review
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Abdo Z, Schütte UME, Bent SJ, Williams CJ, Forney LJ, Joyce P (2006) Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes. Environ Microbiol 8:929–938

    Article  PubMed  Google Scholar 

  • Acinas SG, Sarma-Ruptavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Appl Environ Microbiol 71:8966–8969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779

    Article  CAS  PubMed  Google Scholar 

  • Avis PG, Dickie IA, Mueller GM (2006) A ‘dirty’ business: testing limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi. Mol Ecol 15:873–882

    Article  CAS  PubMed  Google Scholar 

  • Becker R, Böger P, Oehlmann R, Ernst A (2000) PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities. Appl Environ Microbiol 66:4945–4953

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Behr S, Mätzig M, Levin A, Eickhoff H, Heller C (1999) A fully automated multicapillary electrophoresis device for DNA analysis. Electrophoresis 20:1492–1507

    Article  CAS  PubMed  Google Scholar 

  • Bent SJ, Forney LJ (2008) The tragedy of the uncommon: understanding limitations in the analysis of microbial diversity. ISME J 0:1–7

    Google Scholar 

  • Berg G, Krechel A, Ditz M, Sikora RA, Ulrich A, Hallmann J (2005) Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi. FEMS Microbiol Ecol 51:215–229

    Article  CAS  PubMed  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 

  • Blackwood CB, Buyer JS (2007) Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities. Soil Biol Biochem 39:590–599

    Article  CAS  Google Scholar 

  • Blackwood CB, Marsh T, Kim SH, Paul EA (2003) Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities. Appl Environ Microbiol 69:926–932

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blackwood CB, Hudleston D, Zak DR, Buyer JS (2007) Interpreting ecological diversity indices applied to terminal restriction fragment length polymorphism data: insights from simulated microbial communities. Appl Environ Microbiol 73:5276–5283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brown MV, Schwalbach MS, Hewson I, Fuhrman JA (2005) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Environ Microbiol 7:1466–1479

    Article  CAS  PubMed  Google Scholar 

  • Cao Y, Cherr GN, Córdova-Kreylos AL, Fan TW-M, Green PG, Higashi RM, LaMontagne MG, Scow KM, Vines CA, Yuan J, Holden PA (2006) Relationships between sediment microbial communities and pollutants in two California salt marshes. Microb Ecol 52:619–633

    Article  CAS  PubMed  Google Scholar 

  • Clement BG, Kehl LE, DeBord KL, Kitts CL (1998) Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J Microbiol Methods 31:135–142

    Article  CAS  Google Scholar 

  • Collins RE, Rocap G (2007) REPK: an analytical web server to select restriction endonucleases for terminal restriction fragment length polymorphism analysis. Nucleic Acids Res 35:58–62

    Article  Google Scholar 

  • Crosby LD, Criddle CS (2003) Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. Biotechniques 34:790–764

    Article  CAS  PubMed  Google Scholar 

  • Culman SW, Duxbury JM, Lauren JG, Thies JE (2006) Microbial community response to soil solarization in Nepal's rice–wheat cropping system. Soil Biol Biochem 38:3359–3371

    Article  CAS  Google Scholar 

  • Denaro R, D'Auria G, Di Marco G, Genovese M, Troussellier M, Yakimov MM, Giuliano L (2005) Assessing terminal restriction fragment length polymorphism suitability for the description of bacterial community structure and dynamics in hydrocarbon-polluted marine environments. Environ Microbiol 7:78–87

    Article  CAS  PubMed  Google Scholar 

  • Dickie IA, Xu B, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytol 156:527–535

    Article  CAS  PubMed  Google Scholar 

  • Dollhopf SL, Hashsham SA, Tiedje JM (2001) Interpreting 16S rDNA T-RFLP data: application of self-organizing maps and principal component analysis to describe community dynamics and convergence. Microb Ecol 42:495–505

    Article  CAS  PubMed  Google Scholar 

  • Dorigo U, Volatier L, Humbert JF (2005) Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Water Res 39:2207–2218

    Article  CAS  PubMed  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. Appl Environ Microbiol 67:190–197

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Egert M, Friedrich MW (2003) Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure. Appl Environ Microbiol 69:2555–2562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Egert M, Friedrich MW (2005) Post-amplification Klenow fragment treatment alleviates PCR bias caused by partially single-stranded amplicons. J Microbiol Methods 61:69–75

    Article  CAS  PubMed  Google Scholar 

  • Engebretson JJ, Moyer CL (2003) Fidelity of select restriction endonucleases in determining microbial diversity by terminal restriction fragment length polymorphism. Appl Environ Microbiol 69:4823–4829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fedi S, Tremaroli V, Scala D, Perez-Jimenez JR, Fava F, Young L, Zannoni D (2005) T-RFLP analysis of bacterial communities in cyclodextrin-amended bioreactors developed for biodegradation of polychlorinated biphenyls. Res Microbiol 156:201–210

    Article  CAS  PubMed  Google Scholar 

  • Fisher MM, Triplett EW (1999) Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl Environ Microbiol 65:4630–4636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Franklin RB, Mills AL (2003) Multi-scale variation in spatial heterogeneity for microbial community structure in an eastern Virginia agricultural field. FEMS Microbiol Ecol 44:335–346

    Article  CAS  PubMed  Google Scholar 

  • Gauch HG Jr (1992) Statistical analysis of regional yield trials: AMMI analysis of factorial designs. Elsevier, Amsterdam, the Netherlands

    Google Scholar 

  • Genney DR, Anderson IC, Alexander IJ (2006) Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium. New Phytol 170:381–390

    Article  PubMed  Google Scholar 

  • Grant A, Ogilvie LA (2003) Terminal restriction fragment length polymorphism data analysis. Appl Environ Microbiol 69:6342–6343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grüter D, Schmid B, Brandl H (2006) Influence of plant diversity and elevated atmospheric carbon dioxide levels on belowground bacterial diversity. BMC Microbiol 6:68–74

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hahn M, Wilhelm J, Pingoud A (2001) Influence of fluorophore dye labels on the migration behavior of polymerase chain reaction-amplified short tandem repeats during denaturing capillary electrophoresis. Electrophoresis 22:2691–2700

    Article  CAS  PubMed  Google Scholar 

  • Hartmann M, Enkerli J, Widmer F (2007) Residual polymerase activity-induced bias in terminal restriction fragment length polymorphism analysis. Environ Microbiol 9:555–559

    Article  CAS  PubMed  Google Scholar 

  • Hewson I, Fuhrman JA (2006) Improved strategy for comparing microbial assemblage fingerprints. Microb Ecol 51:147–153

    Article  PubMed  Google Scholar 

  • Horz HP, Rotthauwe JH, Lukow T, Liesack W (2000) Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products. J Microbiol Methods 39:197–204

    Article  CAS  PubMed  Google Scholar 

  • Horz HP, Yimga MT, Liesack W (2001) Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling. Appl Environ Microbiol 67:4177–4185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hoshino T, Terahara T, Yamada K, Okuda H, Suzuki I, Tsuneda S, Hirata A, Inamori Y (2006) Long-term monitoring of the succession of a microbial community in activated sludge from a circulation flush toilet as a closed system. FEMS Microbiol Ecol 55:459–470

    Article  CAS  PubMed  Google Scholar 

  • Hullar MAJ, Kaplan LA, Stahl DA (2006) Recurring seasonal dynamics of microbial communities in stream habits. Appl Environ Microbiol 72:713–722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Johnson DE (1998) Applied multivariate methods for data analysts. Brooks/Cole, Pacific Grove, CA, USA

    Google Scholar 

  • Johnson D, Vandenkoornhuyse PJ, Leake JR, Gilbert L, Booth RE, Grime JP, Young JPW, Read DJ (2004) Plant communities affect arbuscular mycorrhizal fungal diversity and community composition in grassland microcosms. New Phytol 161:503–515

    Article  PubMed  Google Scholar 

  • Kanagawa T (2003) Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng 96:317–323

    Article  CAS  PubMed  Google Scholar 

  • Kaplan CW, Kitts CL (2003) Variation between observed and true terminal restriction fragment length is dependent on true TRF length and purine content. J Microbiol Methods 54:121–125

    Article  CAS  PubMed  Google Scholar 

  • Katsivela E, Moore ERB, Maroukli D, Strömpl C, Pieper D, Kalogerakis N (2005) Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites. Biodegradation 16:169–180

    Article  CAS  PubMed  Google Scholar 

  • Kennedy N, Edwards S, Clipson N (2005) Soil bacterial and fungal community structure across a range of unimproved and semi-improved upland grasslands. Microb Ecol 50:463–473

    Article  PubMed  Google Scholar 

  • Kent AD, Smith DJ, Benson BJ, Triplett EW (2003) Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities. Appl Environ Microbiol 69:6768–6776

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kitts CL (2001) Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Intest Microbiol 2:17–25

    CAS  PubMed  Google Scholar 

  • Kohonen T (2000) Self organizing maps. In: Huang TS, Kohonen T (eds) Information science. Springer, Berlin, Heidelberg

    Google Scholar 

  • Kotsyurbenko OR, Chin KJ, Glagolev MV, Stubner S, Simankova MV, Nozhevnikova AN, Conrad R (2004) Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog. Environ Microbiol 6:1159–1173

    Article  CAS  PubMed  Google Scholar 

  • Kurata S, Kanagawa T, Magariyama Y, Takatsu K, Yamada K, Yokomaku T, Kamagata Y (2004) Reevaluation and reduction of a PCR bias caused by reannealing of templates. Appl Environ Microbiol 70:7545–7549

    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 

  • Leckie SE (2005) Methods of microbial community profiling and their application to forest soils. For Ecol Manag 220:88–106

    Article  Google Scholar 

  • Legendre P, Anderson MJ (1999) Distance-based redundancy analysis: testing multispecies responses in multifactorial ecological experiments. Ecol Monogr 69:1–24

    Article  Google Scholar 

  • Legendre P, Gallagher ED (2001) Ecologically meaningful transformations for ordination of species data. Oecologia 129:271–280

    Article  PubMed  Google Scholar 

  • Leybo AI, Netrusov AI, Conrad R (2006) Effect of hydrogen concentration on the community structure of hydrogenotrophic methanogens studied by T-RFLP analysis of 16S rRNA gene amplicons. Microbiology 75:683–688

    Article  CAS  Google Scholar 

  • 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 63:4516–4522

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lord NS, Kaplan CW, Shank P, Kitts CL, Elrod SL (2002) Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions. FEMS Microbiol Ecol 42:327–337

    Article  CAS  PubMed  Google Scholar 

  • Lu Y, Lueders T, Friedrich MW, Conrad R (2005) Detecting active methanogenic populations on rice roots using stable isotope probing. Environ Microbiol 7:326–336

    Article  CAS  PubMed  Google Scholar 

  • Lueders T, Friedrich MW (2003) Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol 69:320–326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lukow T, Dunfield PF, Liesack W (2000) Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiol Ecol 3:241–247

    Article  Google Scholar 

  • Magalhaes C, Bano N, Wiebe WJ, Bordalo AA, Hollibaugh JT (2008) Dynamics of nitrous oxide reductase genes (nosZ) in intertidal rocky biofilms and sediments of the Douro river estuary (Portugal), and their relation to N-biogeochemistry. Microb Ecol 55:259–269

    Article  CAS  PubMed  Google Scholar 

  • Marsh TL (1999) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr Opin Microbiol 2:323–327

    Article  CAS  PubMed  Google Scholar 

  • Marsh TL (2005) Culture-independent microbial community analysis with terminal restriction fragment length polymorphism. Methods Enzymol 397:308–329

    Article  CAS  PubMed  Google Scholar 

  • Marsh TL, Saxman P, Cole J, Tiedje J (2000) Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl Environ Microbiol 66:3616–3620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mintie AT, Heichen RS, Cromack Jr K, Myrold DD, Bottomley PJ (2003) Ammonia-oxidizing bacteria along meadow-to-forest transects in the Oregon Cascade mountains. Appl Environ Microbiol 69:3129–3136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moeseneder MM, Arrieta JM, Muyzer G, Winter C, Herndl G (1999) Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl Environ Microbiol 65:3518–3525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moeseneder MM, Winter C, Arrieta JM, Herndl GJ (2001) Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes. J Microbiol Methods 44:159–172

    Article  CAS  PubMed  Google Scholar 

  • Mohanty SR, Bodelier PLE, Floris V, Conrad R (2006) Differential effects of nitrogenous fertilizers on methane-consuming microbes in rice field and forest soils. Appl Environ Microbiol 72:1346–1354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Moyer CL, Tiedje JM, Dobbs FC, Karl DM (1996) A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. Appl Environ Microbiol 62:2501–2507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mummey DL, Stahl PD (2003) Spatial and temporal variability of bacterial 16S rDNA-based T-RFLP patterns derived from soil of two Wyoming grassland ecosystems. FEMS Microbiol Ecol 46:113–120

    Article  CAS  PubMed  Google Scholar 

  • 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 59:695–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nilsson WB, Strom MS (2002) Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Dis Aquat Org 48:175–185

    Article  CAS  Google Scholar 

  • Noll M, Matthies D, Frenzel P, Derakshani M, Liesack W (2005) Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ Microbiol 7:382–395

    Article  CAS  PubMed  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 

  • Osborne CA, Galic M, Sangwan P, Janssen PH (2005) PCR-generated artifact from 16S rRNA gene-specific primers. FEMS Microbiol Lett 248:183–187

    Article  CAS  PubMed  Google Scholar 

  • Osborne CA, Rees GN, Bernstein Y, Janssen PH (2006) New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities. Appl Environ Microbiol 72:1270–1278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pereira MG, Latchford JW, Mudge SM (2006) The use of terminal restriction fragment length polymorphism (T-RFLP) for the characterization of microbial communities in marine sediments. Geomicrobiol J 23:247–251

    Article  CAS  Google Scholar 

  • Pérez-Jiménez JR, Kerkhof LJ (2005) Phylogeography of sulfate-reducing bacteria among disturbed sediments, disclosed analysis of the dissimilatory sulfite reductase genes (dsrAB). Appl Environ Microbiol 71:1004–1011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pérez-Piqueres A, Edel-Herrmann V, Alabouvette C, Steinberg C (2006) Response of soil microbial communities to compost amendments. Soil Biol Biochem 38:460–470

    Article  CAS  Google Scholar 

  • Pesaro M, Nicollier G, Zeyer J, Widmer F (2004) Impact of soil drying-rewetting stress on microbial communities and activities and on degradation of two crop protection products. Appl Environ Microbiol 70:2577–2587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pett-Ridge J, Firestone MK (2005) Redox fluctuation structures microbial communities in a wet tropical soil. Appl Environ Microbiol 71:6998–7007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Polymenakou PN, Bertilsson S, Tselepides A, Stephanou EG (2005) Links between geographic location, environmental factors, and microbial community composition in sediments of the eastern Mediterranean Sea. Microb Ecol 49:367–378

    Article  CAS  PubMed  Google Scholar 

  • Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64:3724–3730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qiu X, Wu L, Huang H, McDonel PE, Palumbo AV, Tiedje JM, Zhou J (2001) Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Appl Environ Microbiol 67:880–887

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Raftery AE, Newton MA, Satagopan JM, Krivitsky PN (2006) Estimating the integrated likelihood via posterior simulation using the harmonic mean identity. Bayesian Stat 8:1–45

    Google Scholar 

  • Ramakrishnan B, Lueders T, Conrad R, Friedrich M (2000) Effect of soil aggregate size on methanogenesis and archaeal community structure in anoxic rice field soil. FEMS Microbiol Ecol 32:261–270

    Article  CAS  PubMed  Google Scholar 

  • Ramette A (2007) Multivariate analyses in microbial ecology. FEMS Microbiol Ecol 62:142–160

    Article  CAS  PubMed  Google Scholar 

  • Rappe MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394

    Article  CAS  PubMed  Google Scholar 

  • Rasche F, Hödl V, Poll C, Kandeler E, Gerzabek MH, van Elsas JD, Sessitsch A (2006) Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure. FEMS Microbiol Ecol 56:219–235

    Article  CAS  PubMed  Google Scholar 

  • Rees GN, Baldwin DS, Watson GO, Perryman S, Nielsen DL (2004) Ordination and significance testing of microbial community composition derived from terminal restriction fragment length polymorphisms: application of multivariate statistics. Antonie van Leeuwenhoek 86:339–347

    Article  PubMed  Google Scholar 

  • Reysenbach AL, Giver LJ, Wickham GS, Pace NR (1992) Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol 58:3417–3418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rich JJ, Heichen RS, Bottomley PJ, Cromack Jr K, Myrold DD (2003) Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils. Appl Environ Microbiol 69:5974–5982

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ricke P, Kolb S, Braker G (2005) Application of a newly developed ARB software-integrated tool for in silico terminal restriction fragment length polymorphism analysis reveals the dominance of a novel pmoA cluster in a forest soil. Appl Environ Microbiol 71:1671–1673

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rösch C, Bothe H (2004) Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl Environ Microbiol 71:2026–2035

    Article  CAS  Google Scholar 

  • Rösch C, Eilmus S, Bothe H (2006) Approaches to assess the biodiversity of bacteria in natural habitats. Biochem Soc Trans 34:169–173

    Article  PubMed  Google Scholar 

  • Ruan Q, Steele JA, Schwalbach MS, Fuhrman JA, Sun F (2006) A dynamic programming algorithm for binning microbial community profiles. Bioinformatics 22:1508–1514

    Article  CAS  PubMed  Google Scholar 

  • Sait L, Galic M, Strugnell RA, Janssen PH (2003) Secretory antibodies do not affect the composition of the bacterial microbiota in the terminal ileum of 10-week-old mice. Appl Environ Microbiol 69:2100–2109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schmidt M, Priemé A, Stougaard P (2006) Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland. Extremophiles 10:551–562

    Article  CAS  PubMed  Google Scholar 

  • Schwartz E, Adaira KL, Schuur EA (2007) Bacterial community structure correlates with decomposition parameters along a Hawaiian precipitation gradient. Soil Biol Biochem 39:2164–2167

    Article  CAS  Google Scholar 

  • Sessitsch A, Weilharter A, Gerzabek MH, Kirchmann H, Kandeler E (2001) Microbial population structures in soil particle size fractions of a long-term fertilizer field experiment. Appl Environ Microbiol 67:4215–4224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ (2007) MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microb Ecol 53:562–570

    Article  CAS  PubMed  Google Scholar 

  • Singh BK, Munro S, Reid E, Ord B, Potts JM, Paterson E, Millard P (2006a) Investigating microbial community structure in soils by physiological, biochemical, and molecular fingerprinting methods. Eur J Soil Sci 57:72–82

    Article  CAS  Google Scholar 

  • Singh BK, Nazaries L, Munro S, Anderson IC, Campbell DC (2006b) Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community. Appl Environ Microbiol 72:7278–7285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 69:470–479

    Article  CAS  PubMed  Google Scholar 

  • Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62:625–630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tan Z, Hurek T, Reinhold-Hurek B (2003) Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice. Environ Microbiol 5:1009–1015

    Article  CAS  PubMed  Google Scholar 

  • Tang J, Tao J, Urakawa H, Corander J (2007) T-BAPS: a Bayesian statistical tool for comparison of microbial communities using terminal restriction fragment length polymorphism (T-RFLP) data. Stat Appl Genet Mol Biol 6:30

    Article  Google Scholar 

  • Ter Braak CJF (1986) Canonical correspondence analysis: a new eigenvector technique for multivariate direct gradient analysis. Ecology 67:1167–1179

    Article  Google Scholar 

  • Terahara T, Hoshino T, Tsuneda S, Hirata A, Inamori Y (2004) Monitoring the microbial population dynamics at the start-up stage of wastewater treatment reactor by terminal restriction fragment length polymorphism analysis based on 16S rDNA and rRNA gene sequences. J Biosci Bioeng 98:425–428

    Article  CAS  PubMed  Google Scholar 

  • Thies JE (2007) Soil microbial community analysis using terminal restriction fragment length polymorphisms. Soil Sci Soc Am 71:579–591

    Article  CAS  Google Scholar 

  • Thies JE, Holmes AM, Vachor A (2001) Application of molecular techniques to studies in Rhizobium ecology: a review. Aust J Exp Agric 41:299–319

    Article  CAS  Google Scholar 

  • Thies FL, König W, König B (2007) Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprint. J Med Microbiol 56:755–761

    Article  CAS  PubMed  Google Scholar 

  • Tiquia SM, Ichida JM, Keener HM, Elwell DL, Burtt EH, Michel FC (2005) Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes. Appl Microbiol Biotechnol 67:412–419

    Article  CAS  PubMed  Google Scholar 

  • Torsvik V, Øvreas L, Thingstad TF (2002) Prokaryotic diversity—magnitude, dynamics, and controlling factors. Science 35:1064–1066

    Article  Google Scholar 

  • Tu O, Knott T, Marsh M, Bechtol K, Harris D, Barker D, Bashkin J (1998) The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA. Nucleic Acid Res 26:2797–2802

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tullos RE (1997) Assessment of similarity indices for undesirable properties and a new tripartite similarity index based on cost functions. In: Palm ME, Chapela IH (eds) Mycology in sustainable development: expanding concepts, vanishing borders. Parkway, Boone, NC, pp 122–143

    Google Scholar 

  • von Wintzingerode F, Gobel UB, Stackebrandt E (1997) Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 21:213–229

    Article  Google Scholar 

  • Wang GCY, Wang Y (1997) Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. Appl Environ Microbiol 63:4645–4650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang M, Ahrne S, Antonsson M, Molin G (2004) T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages. J Microbiol Methods 59:53–69

    Article  CAS  PubMed  Google Scholar 

  • Weber S, Lueders T, Friedrich MW, Conrad R (2001) Methanogenic populations involved in the degradation of rice straw in anoxic paddy soil. FEMS Microbiol Ecol 38:11–20

    Article  CAS  Google Scholar 

  • Weng L, Rubin EM, Bristow J (2006) Application of sequence-based methods in human microbial ecology. Genome Res 16:316–322

    Article  CAS  PubMed  Google Scholar 

  • Wise MJ, Osborn AM (2001) TRUFFLER: programs to study microbial community composition and flux from fluorescent DNA fingerprinting data. Bioinformatics and Bioengineering Conference, pp 129–135

  • Wu XL, Fiedrich MW, Conrad R (2006) Diversity and ubiquity of thermophilic methanogenic archaea in temperate anoxic soils. Environ Microbiol 8:394–404

    Article  CAS  PubMed  Google Scholar 

  • Young MW (1987) Multidimensional scaling: history, theory, and application. Hamer RM (ed) Erlbaum, Hillsdale, NJ

  • Zhou X, Brown CJ, Abdo Z, Davis CC, Hansmann MA, Joyce P, Foster JA, Forney LJ (2007) Disparity in the vaginal microbial community composition of healthy Caucasian and black woman. ISME J 1:121–133

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Larry J. Forney.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schütte, U.M.E., Abdo, Z., Bent, S.J. et al. Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities. Appl Microbiol Biotechnol 80, 365–380 (2008). https://doi.org/10.1007/s00253-008-1565-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1565-4

Keywords

Navigation