This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Egert, M.
Right arrow Articles by Friedrich, M. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Egert, M.
Right arrow Articles by Friedrich, M. W.
Agricola
Right arrow Articles by Egert, M.
Right arrow Articles by Friedrich, M. W.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, May 2003, p. 2555-2562, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2555-2562.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

Markus Egert and Michael W. Friedrich*

Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg/Lahn, Germany

Received 8 November 2002/ Accepted 5 February 2003

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

* Corresponding author. Mailing address: Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, D-35043 Marburg/Lahn, Germany. Phone: 49-6421-178830. Fax: 49-6421-178809. E-mail: michael.friedrich{at}staff.uni-marburg.de.

Applied and Environmental Microbiology, May 2003, p. 2555-2562, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2555-2562.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rossi, P., Gillet, F., Rohrbach, E., Diaby, N., Holliger, C. (2009). Statistical Assessment of Variability of Terminal Restriction Fragment Length Polymorphism Analysis Applied to Complex Microbial Communities. Appl. Environ. Microbiol. 75: 7268-7270 [Abstract] [Full Text]  
  • Pieper, R., Bindelle, J., Rossnagel, B., Van Kessel, A., Leterme, P. (2009). Effect of Carbohydrate Composition in Barley and Oat Cultivars on Microbial Ecophysiology and Proliferation of Salmonella enterica in an In Vitro Model of the Porcine Gastrointestinal Tract. Appl. Environ. Microbiol. 75: 7006-7016 [Abstract] [Full Text]  
  • Eiler, A., Beier, S., Sawstrom, C., Karlsson, J., Bertilsson, S. (2009). High Ratio of Bacteriochlorophyll Biosynthesis Genes to Chlorophyll Biosynthesis Genes in Bacteria of Humic Lakes. Appl. Environ. Microbiol. 75: 7221-7228 [Abstract] [Full Text]  
  • Hamberger, A., Horn, M. A., Dumont, M. G., Murrell, J. C., Drake, H. L. (2008). Anaerobic Consumers of Monosaccharides in a Moderately Acidic Fen. Appl. Environ. Microbiol. 74: 3112-3120 [Abstract] [Full Text]  
  • Winderl, C., Anneser, B., Griebler, C., Meckenstock, R. U., Lueders, T. (2008). Depth-Resolved Quantification of Anaerobic Toluene Degraders and Aquifer Microbial Community Patterns in Distinct Redox Zones of a Tar Oil Contaminant Plume. Appl. Environ. Microbiol. 74: 792-801 [Abstract] [Full Text]  
  • Dekio, I., Sakamoto, M., Hayashi, H., Amagai, M., Suematsu, M., Benno, Y. (2007). Characterization of skin microbiota in patients with atopic dermatitis and in normal subjects using 16S rRNA gene-based comprehensive analysis. J Med Microbiol 56: 1675-1683 [Abstract] [Full Text]  
  • Thies, F. L., Konig, W., Konig, B. (2007). Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Med Microbiol 56: 755-761 [Abstract] [Full Text]  
  • Eiler, A., Bertilsson, S. (2007). Flavobacteria Blooms in Four Eutrophic Lakes: Linking Population Dynamics of Freshwater Bacterioplankton to Resource Availability. Appl. Environ. Microbiol. 73: 3511-3518 [Abstract] [Full Text]  
  • Thies, J. E. (2007). Soil Microbial Community Analysis using Terminal Restriction Fragment Length Polymorphisms. Soil Sci. 71: 579-591 [Abstract] [Full Text]  
  • Martin, K. J. (2007). Introduction to Molecular Analysis of Ectomycorrhizal Communities. Soil Sci. 71: 601-610 [Abstract] [Full Text]  
  • Gentile, M. E., Jessup, C. M., Nyman, J. L., Criddle, C. S. (2007). Correlation of Functional Instability and Community Dynamics in Denitrifying Dispersed-Growth Reactors. Appl. Environ. Microbiol. 73: 680-690 [Abstract] [Full Text]  
  • Case, R. J., Boucher, Y., Dahllof, I., Holmstrom, C., Doolittle, W. F., Kjelleberg, S. (2007). Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies. Appl. Environ. Microbiol. 73: 278-288 [Abstract] [Full Text]  
  • Hartmann, M., Widmer, F. (2006). Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices. Appl. Environ. Microbiol. 72: 7804-7812 [Abstract] [Full Text]  
  • Rooney, D., Kennedy, N., Deering, L., Gleeson, D., Clipson, N. (2006). Effect of sheep urine deposition on the bacterial community structure in an acidic upland grassland soil.. Appl. Environ. Microbiol. 72: 7231-7237 [Abstract] [Full Text]  
  • Singh, B. K., Nazaries, L., Munro, S., Anderson, I. C., Campbell, C. D. (2006). Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community{triangledown}. Appl. Environ. Microbiol. 72: 7278-7285 [Abstract] [Full Text]  
  • Pang, C. M., Liu, W.-T. (2006). Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure. Appl. Environ. Microbiol. 72: 5702-5712 [Abstract] [Full Text]  
  • Brazelton, W. J., Schrenk, M. O., Kelley, D. S., Baross, J. A. (2006). Methane- and Sulfur-Metabolizing Microbial Communities Dominate the Lost City Hydrothermal Field Ecosystem. Appl. Environ. Microbiol. 72: 6257-6270 [Abstract] [Full Text]  
  • Watrud, L. S., Martin, K., Donegan, K. K., Stone, J. K., Coleman, C. G. (2006). Comparison of taxonomic, colony morphotype and PCR-RFLP methods to characterize microfungal diversity.. Mycologia 98: 384-392 [Abstract] [Full Text]  
  • Lepere, C., Boucher, D., Jardillier, L., Domaizon, I., Debroas, D. (2006). Succession and Regulation Factors of Small Eukaryote Community Composition in a Lacustrine Ecosystem (Lake Pavin). Appl. Environ. Microbiol. 72: 2971-2981 [Abstract] [Full Text]  
  • Langenheder, S., Lindstrom, E. S., Tranvik, L. J. (2006). Structure and Function of Bacterial Communities Emerging from Different Sources under Identical Conditions. Appl. Environ. Microbiol. 72: 212-220 [Abstract] [Full Text]  
  • Hongoh, Y., Deevong, P., Inoue, T., Moriya, S., Trakulnaleamsai, S., Ohkuma, M., Vongkaluang, C., Noparatnaraporn, N., Kudo, T. (2005). Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host. Appl. Environ. Microbiol. 71: 6590-6599 [Abstract] [Full Text]  
  • Egert, M., Stingl, U., Dyhrberg Bruun, L., Pommerenke, B., Brune, A., Friedrich, M. W. (2005). Structure and Topology of Microbial Communities in the Major Gut Compartments of Melolontha melolontha Larvae (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 71: 4556-4566 [Abstract] [Full Text]  
  • Macbeth, T. W., Cummings, D. E., Spring, S., Petzke, L. M., Sorenson, K. S. Jr. (2004). Molecular Characterization of a Dechlorinating Community Resulting from In Situ Biostimulation in a Trichloroethene-Contaminated Deep, Fractured Basalt Aquifer and Comparison to a Derivative Laboratory Culture. Appl. Environ. Microbiol. 70: 7329-7341 [Abstract] [Full Text]  
  • 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 [Abstract] [Full Text]  
  • Nocker, A., Lepo, J. E., Snyder, R. A. (2004). Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm. Appl. Environ. Microbiol. 70: 6834-6845 [Abstract] [Full Text]  
  • 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 [Abstract] [Full Text]  
  • Idris, R., Trifonova, R., Puschenreiter, M., Wenzel, W. W., Sessitsch, A. (2004). Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense. Appl. Environ. Microbiol. 70: 2667-2677 [Abstract] [Full Text]  
  • Pester, M., Friedrich, M. W., Schink, B., Brune, A. (2004). pmoA-Based Analysis of Methanotrophs in a Littoral Lake Sediment Reveals a Diverse and Stable Community in a Dynamic Environment. Appl. Environ. Microbiol. 70: 3138-3142 [Abstract] [Full Text]  
  • Conn, V. M., Franco, C. M. M. (2004). Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones. Appl. Environ. Microbiol. 70: 1787-1794 [Abstract] [Full Text]  
  • Egert, M., Wagner, B., Lemke, T., Brune, A., Friedrich, M. W. (2003). Microbial Community Structure in Midgut and Hindgut of the Humus-Feeding Larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae). Appl. Environ. Microbiol. 69: 6659-6668 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»