Oral Biology pp 139-152 | Cite as

Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE)

  • José F. SiqueiraJr.Email author
  • Mitsuo Sakamoto
  • Alexandre S. Rosado
Part of the Methods in Molecular Biology book series (MIMB, volume 1537)


In their natural environments, microorganisms usually live in organized communities. Profiling analysis of microbial communities has recently assumed special relevance as it allows a thorough understanding of the diversity of the microbiota, its behavior over time, and the establishment of patterns associated with health and disease. The application of molecular biology approaches holds the advantage of including culture-difficult and as-yet-uncultivated phylotypes in the profiles, providing a more comprehensive picture of the microbial community. This chapter focuses on two particular techniques, namely, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE), both of which have been widely used in environmental studies and have been successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.

Key words

Human oral microbiota 16S rRNA gene Terminal restriction fragment length polymorphism (T-RFLP) Denaturing gradient gel electrophoresis (DGGE) 



This work was supported in part by grants from the Brazilian National Research Council (CNPq) and FAPERJ (José Siqueira and Alexandre Rosado) and by a Grant-in-Aid for Scientific Research (No. 13672202) from the Japan Society for the Promotion of Science (Mitsuo Sakamoto). We are grateful to Natalia Franco for drawing the DGGE schemes.


  1. 1.
    Sakamoto M, Takeuchi Y, Umeda M, Ishikawa I, Benno Y (2003) Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis. J Med Microbiol 52:79–89CrossRefPubMedGoogle Scholar
  2. 2.
    Zijnge V, Harmsen HJ, Kleinfelder JW, van der Rest ME, Degener JE, Welling GW (2003) Denaturing gradient gel electrophoresis analysis to study bacterial community structure in pockets of periodontitis patients. Oral Microbiol Immunol 18:59–65CrossRefPubMedGoogle Scholar
  3. 3.
    Sakamoto M, Huang Y, Ohnishi M, Umeda M, Ishikawa I, Benno Y (2004) Changes in oral microbial profiles after periodontal treatment as determined by molecular analysis of 16S rRNA genes. J Med Microbiol 53:563–571CrossRefPubMedGoogle Scholar
  4. 4.
    Siqueira JF Jr, Rôças IN, Rosado AS (2004) Investigation of bacterial communities associated with asymptomatic and symptomatic endodontic infections by denaturing gradient gel electrophoresis fingerprinting approach. Oral Microbiol Immunol 19:363–370CrossRefPubMedGoogle Scholar
  5. 5.
    Rôças IN, Siqueira JF Jr, Aboim MC, Rosado AS (2004) Denaturing gradient gel electrophoresis analysis of bacterial communities associated with failed endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 98:741–749CrossRefPubMedGoogle Scholar
  6. 6.
    Siqueira JF Jr, Rôças IN, Rosado AS (2005) Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of endodontic infections. J Endod 31:775–782CrossRefPubMedGoogle Scholar
  7. 7.
    Li Y, Ku CY, Xu J, Saxena D, Caufield PW (2005) Survey of oral microbial diversity using PCR-based denaturing gradient gel electrophoresis. J Dent Res 84:559–564CrossRefPubMedGoogle Scholar
  8. 8.
    Sakamoto M, Rôças IN, Siqueira JF Jr, Benno Y (2006) Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections. Oral Microbiol Immunol 21:112–122CrossRefPubMedGoogle Scholar
  9. 9.
    Zijnge V, Welling GW, Degener JE, van Winkelhoff AJ, Abbas F, Harmsen HJ (2006) Denaturing gradient gel electrophoresis as a diagnostic tool in periodontal microbiology. J Clin Microbiol 44:3628–3633CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Machado de Oliveira JC, Siqueira JF Jr, Rôças IN, Baumgartner JC, Xia T, Peixoto RS, Rosado AS (2007) Bacterial community profiles of endodontic abscesses from Brazilian and USA subjects as compared by denaturing gradient gel electrophoresis analysis. Oral Microbiol Immunol 22:14–18CrossRefPubMedGoogle Scholar
  11. 11.
    Siqueira JF Jr, Rôças IN, Debelian GJ, Carmo FL, Paiva SS, Alves FR, Rosado AS (2008) Profiling of root canal bacterial communities associated with chronic apical periodontitis from Brazilian and Norwegian subjects. J Endod 34:1457–1461CrossRefPubMedGoogle Scholar
  12. 12.
    Alves FR, Siqueira JF Jr, Carmo FL, Santos AL, Peixoto RS, Rôças IN, Rosado AS (2009) Bacterial community profiling of cryogenically ground samples from the apical and coronal root segments of teeth with apical periodontitis. J Endod 35:486–492CrossRefPubMedGoogle Scholar
  13. 13.
    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 63:4516–4522PubMedPubMedCentralGoogle Scholar
  14. 14.
    Clement BG, Kehl LE, De Bord KL, Kitts CL (1998) Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J Microbiol Methods 31:135–142CrossRefGoogle Scholar
  15. 15.
    Spiegelman D, Whissell G, Greer CW (2005) A survey of the methods for the characterization of microbial consortia and communities. Can J Microbiol 51:355–386CrossRefPubMedGoogle Scholar
  16. 16.
    Matsumoto M, Sakamoto M, Hayashi H, Benno Y (2005) Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota. J Microbiol Methods 61:305–319CrossRefPubMedGoogle Scholar
  17. 17.
    Sakamoto M, Umeda M, Benno Y (2005) Molecular analysis of human oral microbiota. J Periodontal Res 40:277–285CrossRefPubMedGoogle Scholar
  18. 18.
    Gasser RB (1998) What’s in that band? Int J Parasitol 28:989–996CrossRefPubMedGoogle Scholar
  19. 19.
    Myers RM, Fischer SG, Lerman LS, Maniatis T (1985) Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131–3145CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    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 59:695–700PubMedPubMedCentralGoogle Scholar
  21. 21.
    Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73:127–141CrossRefPubMedGoogle Scholar
  22. 22.
    Machado de Oliveira JC, Gama TG, Siqueira JF Jr, Rôças IN, Peixoto RS, Rosado AS (2007) On the use of denaturing gradient gel electrophoresis approach for bacterial identification in endodontic infections. Clin Oral Investig 11:127–132CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • José F. SiqueiraJr.
    • 1
    • 2
    Email author
  • Mitsuo Sakamoto
    • 3
  • Alexandre S. Rosado
    • 4
  1. 1.Molecular Microbiology Laboratory, Department of EndodonticsEstácio de Sá UniversityRio de JaneiroBrazil
  2. 2.Faculty of DentistryEstácio de Sá UniversityRio de JaneiroBrazil
  3. 3.Microbe Division/Japan Collection of MicroorganismsRIKEN BioResource CenterWakoJapan
  4. 4.Institute of Microbiology Prof. Paulo de GóesFederal University of Rio de JaneiroRio de JaneiroBrazil

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