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Updated: Jun 10, 2026

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Microbial community profiling using terminal restriction fragment length polymorphism (T-RFLP) and denaturing
José F Siqueira1, Mitsuo Sakamoto, Alexandre S Rosado
1Molecular Microbiology Laboratory, Department of Endodontics, Faculty of Dentistry, Estácio de Sá University, Rio de Janeiro, RJ, Brazil.
Abstract:
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: the 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 recently successfully used by the authors in the study of the oral microbial communities associated with conditions of health and disease.
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