Abstract
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a method for rapid profiling of mixed populations of an homologous amplicon (i.e., diverse sequences of a single gene). It combines restriction fragment analysis of a PCR-amplified gene marker with automated sequencing gel technology. One primer used in PCR amplification of the marker gene is labeled at the 5′ terminus with a fluorescent dye, in order that the terminal restriction fragments (T-RFs) of the digested amplicon can be detected and quantified (1–3). Detailed evaluations of T-RFLP analysis have shown that, in most cases, both the sizes and relative signal intensities of the individual T-RFs in a sample are highly reproducible. Consequently, T-RFLP analysis is an excellent tool for rapidly comparing microbial communities (4–6).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Liu, W. T., Marsh, T. L., Cheng, H., and Forney, L. J. (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516–4522.
Clement, B. G., Kehl, L. E., DeBord, K. L., and Kitts, C. L. (1998) Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J. Microbiol. Methods 31, 135–142.
Marsh, T. L. (1999) Terminal restriction fragment length polymorphism (TRFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr. Opin. Microbiol. 2, 323–327.
Osborn, A. M., Moore, E. R. B., and Timmis, K. N. (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.
Lukow, T., Dunfield, P. F., and Liesack, W. (2000) Use of the T-RFLP analysis to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microb. Ecol. 32, 241–247.
Dollhopf, S. L., Hashsham, S. A., and Tiedje, J. M. (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.
Braker, G., Ayala-del-Rio, H. L., Devol, A. H., Fesefeldt, A., and Tiedje, J. M. (2001) Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in pacific northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes. Appl. Environ. Microbiol. 67, 1893–1901.
Buckley, D. H. and Schmidt, T. M. (2001) The structure of microbial communities in soil and the lasting impact of cultivation. Microb. Ecol. 42, 11–21.
Covert, J. S. and Moran, M. A. (2001) Molecular characterization of estuarine bacterial communities that use high-and low-molecular weight fractions of dissolved organic carbon. Aquat. Microb. Ecol. 25, 127–139.
Dunbar, J., Ticknor, L. O., and Kuske, C. R. (2000) Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66, 2943–2950.
Flynn, S. J., Loffler, F. E., and Tiedje, J. M. (2000) Microbial community changes associated with a shift from reductive dechlorination of PCE to reductive dechlorination of cis-DCE and VC. Environ. Sci. Technol. 34, 1056–1061.
Garland, J. L., Cook, K. L., Adams, J. L., and Kerkhof, L. (2001) Culturability as an indicator of succession in microbial communities. Microb. Ecol. 42, 150–158.
Guieysse, B., Wikström, P., Forsman, M., and Mattiasson, B. (2001) Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor. Appl. Microbiol. Biotechnol. 56, 780–787.
Hiraishi, A., Iwasaki, M., and Shinjo, H. (2000) Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge. J. Bioscience & Bioengineering 90, 148–156.
Leser, T. D., Lindecrona, R. H., Jensen, T. K., Jensen, B. B., and Møller, K. (2000) Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl. Environ. Microbiol. 66, 3290–3296.
Liu, W. T., Marsh, T. L., and Forney, L. J. (1998) Determination of the microbial diversity of anaerobic-aerobic activated sludge by a novel molecular biological technique. Water Sci. Technol. 37, 417–422.
Lüdemann, H., Arth, I., and Liesack, W. (2000) Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Appl. Environ. Microbiol. 66, 754–762.
Moeseneder, M. M., Arrieta, J. M., Muyzer, G., Winter, C., and Herndl, G. J. (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.
Sessitsch, A., Weilharter, A., Gerzabek, M. H., Kirchmann, H., and Kandeler, E. (2001) Microbial population structures in soil particle size fractions of a long-term fertilizer field experiment. Appl. Environ. Microbiol. 67, 4215–4224.
Urakawa, H., Yoshida, T., Nishimura, M., and Ohwada, K. (2000) Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling. Environ. Microbiol. 2, 542–554.
Chin, K. J., Lukow, T., Stubner, S., and Conrad, R. (1999) Structure and function of the methanogenic archaeal community in stable cellulose-degrading enrichment cultures at two different temperatures (15 and 30°C). FEMS Microbiol. Ecol. 30, 313–326.
Chin, K.-J., Lukow, T., and Conrad, R. (1999) Effect of temperature on structure and function of the methanogenic archaeal community in an anoxic rice field soil. Appl. Environ. Microbiol. 65, 2341–2349.
Fey, A., Chin, K.-J., and Conrad, R. (2001) Thermophilic methanogens in rice field soil. Environ. Microbiol. 3, 295–303.
Friedrich, M. W., Schmitt-Wagner, D., Lueders, T., and Brune, A. (2001) Axial differences in community structure of Crenarchaeota and Euryarchaeota in the highly compartmentalized gut of the soil-feeding termite Cubitermes orthognathus. Appl. Environ. Microbiol. 67, 4880–4890.
Lueders, T. and Friedrich, M. (2000) Archaeal population dynamics during sequential reduction processes in rice field soil. Appl. Environ. Microbiol. 66, 2732–2742.
Lueders, T. and Friedrich, M. (2002) Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil. Appl. Environ. Microbiol. 68, 2484–2494.
Moeseneder, M. M., Winter, C., Arrieta, J. M., and Herndl, G. J. (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.
Nüsslein, B., Chin, K. J., Eckert, W., and Conrad, R. (2001) Evidence for anaerobic syntrophic acetate oxidation during methane production in the profundal sediment of subtropical Lake Kinneret (Israel). Environ. Microbiol. 3, 460–470.
Ramakrishnan, B., Lueders, T., Dunfield, P. F., Conrad, R., and Friedrich, M. W. (2001) Archaeal community structures in rice soil from different geographical regions before and after initiation of methane production. FEMS Microbiol. Ecol. 37, 175–186.
Takai, K., Moser, D. P., DeFlaun, M., Onstott, T. C., and Fredrickson, J. K. (2001) Archaeal diversity in waters from deep South African gold mines. Appl. Environ. Microbiol. 67, 5750–5760.
Weber, S., Lueders, T., Friedrich, M. W., and Conrad, R. (2001) Methanogenic populations involved in the degradation of rice straw in anoxic paddy soil. FEMS Microbiol. Ecol. 38, 11–20.
Derakshani, M., Lukow, T., and Liesack, W. (2001) Novel bacterial lineages at the (sub)division level as detected by signature nucleotide-targeted recovery of 16S rRNA genes from bulk soil and rice roots of flooded rice microcosms. Appl. Environ. Microbiol. 67, 623–631.
Horswell, J., Cordiner, S. J., Maas, E. W., Martin, T. M., Sutherland, B. W., Speir, T. W., et al. (2002) Forensic comparison of soils by bacterial community DNA profiling. J. Forensic Sci. 47, 350–353.
Giraffa, G. and Neviani, E. (2001) DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems. Int. J. Food Microbiol. 67, 19–34.
Regan, J. M., Harrington, G. W., and Noguera, D. R. (2002) Ammonia-and nitrite-oxidizing bacterial communities in a pilot-scale chloraminated drinking water distribution system. Appl. Environ. Microbiol. 68, 73–81.
Horz, H.-P., Rotthauwe, J. H., Lukow, T., and 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.
Scala, D. J. and Kerkhof, L. J. (2000) Horizontal heterogeneity of denitrifying bacterial communities in marine sediments by terminal restriction fragment length polymorphism analysis. Appl. Environ. Microbiol. 66, 1980–1986.
Horz, H.-P., Yimga, M. T., and 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.
Lueders, T., Chin, K.-J., Conrad, R., and Friedrich, M. (2001) Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage. Environ. Microbiol. 3, 194–204.
Bruce, K. D. (1997) Analysis of mer gene subclasses within bacterial communities in soils and sediments resolved by fluorescent-PCR-restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 63, 4914–4919.
Dunbar, J., Ticknor, L. O., and Kuske, C. R. (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.
Suzuki, M. T. and Giovannoni, S. J. (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625–630.
v. Wintzingerode, F., Göbel, U. B., and Stackebrandt, E. (1997) Determination of microbial diversity in environmental samples—pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213–229.
Legendre, P. and Legendre, L. (1998). Numerical Ecology, second English edition. Elsevier, Amsterdam, The Netherlands.
Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker, C. T., Saxman, P. R., Stredwick, J. M., et al. (2000) The RDP (Ribosomal Database Project) continues. Nucl. Acids Res. 28, 173–174.
Akkermans, A. D. L., van Elsas, J. D., and de Bruijn, F. J., eds. (1995) Molecular Microbial Ecology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands.
Hurt, R. A., Qiu, X. Y., Wu, L. Y., Roh, Y., Palumbo, A. V., Tiedje, J. M., et al. (2001) Simultaneous recovery of RNA and DNA from soils and sediments. Appl. Environ. Microbiol. 67, 4495–4503.
Miller, D. N., Bryant, J. E., Madsen, E. L., and Ghiorse, W. C. (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol. 65, 4715–4724.
Krsek, M. and Wellington, E. M. H. (1999) Comparison of different methods for the isolation and purification of total community DNA from soil. J. Microbiol. Methods 39, 1–16.
Griffiths, R. I., Whiteley, A. S., O’Donnell, A. G., and Bailey, M. J. (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA-and rRNA-based microbial community composition. Appl. Environ. Microbiol. 66, 5488–5491.
Frostegård, A., Courtois, S., Ramisse, V., Clerc, S., Bernillon, D., LeGall, F., et al. (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl. Environ. Microbiol. 65, 5409–5420.
Kowalchuk, G. A., Naoumenko, Z. S., Derikx, P. J. L., Felske, A., Stephen, J. R., and Arkhipchenko, I. A. (1999) Molecular analysis of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in compost and composted materials. Appl. Environ. Microbiol. 65, 396–403.
Weber, S., Stubner, S., and Conrad, R. (2001) Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microbiol. 67, 1318–1327.
Liu, W. T., Linning, K. D., Nakamura, K., Mino, T., Matsuo, T., and Forney, L. J. (2000) Microbial community changes in biological phosphate-removal systems on altering sludge phosphorus content. Microbiology 146, 1099–1107.
Schwieger, F. and Tebbe, C. C. (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol. 64, 4870–4876.
Peters, S., Koschinsky, S., Schwieger, F., and Tebbe, C. C. (2000) Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66, 930–936.
Marsh, T. L., Saxman, P., Cole, J., and 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.
Egert, M. and Friedrich, M. W. (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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Liesack, W., Dunfield, P.F. (2004). T-RFLP Analysis. In: Walker, J.M., Spencer, J.F.T., Ragout de Spencer, A.L. (eds) Environmental Microbiology. Methods in Biotechnology, vol 16. Humana Press. https://doi.org/10.1385/1-59259-765-3:023
Download citation
DOI: https://doi.org/10.1385/1-59259-765-3:023
Publisher Name: Humana Press
Print ISBN: 978-1-58829-116-5
Online ISBN: 978-1-59259-765-9
eBook Packages: Springer Protocols