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Coupling of denaturing high-performance liquid chromatography and terminal restriction fragment length polymorphism with precise fragment sizing for microbial community profiling and characterization
- Source :
- Applied and Environmental Microbiology, Applied and Environmental Microbiology, American Society for Microbiology, 2010, 76 (3), pp.648-51. ⟨10.1128/AEM.01556-09⟩
- Publication Year :
- 2010
- Publisher :
- HAL CCSD, 2010.
-
Abstract
- Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.
- Subjects :
- DNA, Bacterial
Computational biology
DNA, Ribosomal
Models, Biological
Applied Microbiology and Biotechnology
DNA sequencing
Restriction fragment
Denaturing high performance liquid chromatography
03 medical and health sciences
RNA, Ribosomal, 16S
Methods
[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology
Chromatography, High Pressure Liquid
Ecosystem
Phylogeny
Fluorescent Dyes
030304 developmental biology
Genetics
0303 health sciences
Bacteria
Models, Genetic
Ecology
biology
030306 microbiology
Computational Biology
Genetic Variation
Biodiversity
Sequence Analysis, DNA
DNA Fingerprinting
Terminal restriction fragment length polymorphism
DNA profiling
Genes, Bacterial
biology.protein
Nucleic Acid Conformation
Amplified fragment length polymorphism
Restriction fragment length polymorphism
Community Fingerprinting
Polymorphism, Restriction Fragment Length
Food Science
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 00992240 and 10985336
- Database :
- OpenAIRE
- Journal :
- Applied and Environmental Microbiology, Applied and Environmental Microbiology, American Society for Microbiology, 2010, 76 (3), pp.648-51. ⟨10.1128/AEM.01556-09⟩
- Accession number :
- edsair.doi.dedup.....cb3e8ae03645c72034ecec5b9644d27b