Skip to main content
SpringerLink
Log in

Terminal Restriction Fragment Length Polymorphism (T-RFLP) Profiling of Bacterial 16S rRNA Genes

  • Protocol
  • First Online:
Environmental Microbiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1096))

Abstract

T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacterial communities.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Liu W-T et al (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63:4516–4522

    CAS  PubMed Central  PubMed  Google Scholar 

  2. Clement BG et al (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 

  3. Marsh TL (1999) Terminal restriction 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 

  4. 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 

  5. Moeseneder MM et al (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

    CAS  PubMed Central  PubMed  Google Scholar 

  6. 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 

  7. Brodie EL, Edwards S, Clipson N (2002) Bacterial community dynamics across a floristic gradient in a temperate upland grassland ecosystem. Microb Ecol 44:260–270

    Article  CAS  PubMed  Google Scholar 

  8. 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 Central  PubMed  Google Scholar 

  9. Hartmann M et al (2005) Semi-automated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches. J Microbiol Methods 61:349–360

    Article  CAS  PubMed  Google Scholar 

  10. Orcutt B et al (2009) An interlaboratory comparison of 16S rRNA gene-based terminal restriction fragment length polymorphism and sequencing methods for assessing microbial diversity of seafloor basalts. Environ Microbiol 11:1728–1735

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Junier P, Junier T, Witzel K-P (2008) TRiFLe, a program for in silico terminal restriction fragment length polymorphism analysis with user-defined sequence sets. Appl Environ Microbiol 74:6452–6456

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Roberts DM et al (2012) Directed terminal restriction analysis tool (DRAT): an aid to enzyme selection for directed terminal-restriction fragment length polymorphisms. Methods Ecol Evol 3:24–28

    Article  Google Scholar 

  13. Rösch C, Bothe H (2005) 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  PubMed Central  PubMed  Google Scholar 

  14. Osborne CA et al (2006) New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex communities. Appl Environ Microbiol 72:1270–1278

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Osborne CA, Peoples MB, Janssen PH (2010) Detection of a reproducible, single-member shift in soil bacterial communities exposed to low levels of hydrogen. Appl Environ Microbiol 76:1471–1479

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. 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 Central  PubMed  Google Scholar 

  17. Carson JK et al (2010) Low pore connectivity increases bacterial diversity in soil. Appl Environ Microbiol 76:3936–3942

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Bennett LT, Kasel S, Tibbits J (2009) Woodland trees modulate soil resources and conserve fungal diversity in fragmented landscapes. Soil Biol Biochem 41:2162–2169

    Article  CAS  Google Scholar 

  19. Singh BK et al (2006) 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 Central  PubMed  Google Scholar 

  20. Bissett A et al (2011) Long-term land use effects on soil microbial community structure and function. Appl Soil Ecol 51:66–78

    Article  Google Scholar 

  21. Griffiths RI et al (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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Osborne CA et al (2005) PCR-generated artefact from 16S rRNA gene-specific primers. FEMS Microbiol Lett 248:183–187

    Article  CAS  PubMed  Google Scholar 

  23. Tanner MA et al (1998) Specific ribosomal DNA sequences from diverse environmental settings correlate with experimental contaminants. Appl Environ Microbiol 64:3110–3113

    CAS  PubMed Central  PubMed  Google Scholar 

  24. Zehr JP et al (2003) Nitrogenase genes in PCR and RT-PCR reagents and implications for studies of diverse functional genes. Biotechniques 35:996–1005

    CAS  PubMed  Google Scholar 

  25. 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 Central  PubMed  Google Scholar 

  26. Culman S et al (2009) T-REX: software for the processing and analysis of T-RFLP data. BMC Bioinformatics 10:171

    Article  PubMed Central  PubMed  Google Scholar 

  27. Fitzjohn RG, Dickie IA (2007) TRAMPR: an R package for analysis and matching of terminal-restriction fragment length polymorphism (TRFLP) profiles. Mol Ecol Notes 7:583–587

    Article  CAS  Google Scholar 

  28. Oksanen J et al (2010) vegan: community ecology package. R package version 1.17-3. http://CRAN.R-project.org/package=vegan

  29. Dunbar J, Ticknor LO, Kuske CR (2000) Assessment of microbial diversity in four southwestern United States soil by 16S rRNA gene terminal restriction fragment analysis. Appl Environ Microbiol 66:2943–2950

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Bent SJ et al (2007) Measuring species richness based on microbial community fingerprints: the emperor has no clothes. Appl Environ Microbiol 73:2399–2401

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgement

The author is supported by the Centre for Water Sensitive Cities in the Department of Civil Engineering at Monash University, Clayton, Victoria, Australia.

Author information

Authors and Affiliations

  1. Department of Civil Engineering, Centre for Water Sensitive Cities, Monash University, Clayton, VIC, Australia

    Catherine A. Osborne

Authors
  1. Catherine A. Osborne
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Dept. Chemistry & Biomolecular Sciences, Macquarie University, Sydney, New South Wales, Australia

    Ian T. Paulsen

  2. School of Molecular Bioscience, University of Sydney, Camperdown, New South Wales, Australia

    Andrew J. Holmes

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Osborne, C.A. (2014). Terminal Restriction Fragment Length Polymorphism (T-RFLP) Profiling of Bacterial 16S rRNA Genes. In: Paulsen, I., Holmes, A. (eds) Environmental Microbiology. Methods in Molecular Biology, vol 1096. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-712-9_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-712-9_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-711-2

  • Online ISBN: 978-1-62703-712-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation