Comparisons of Different Hypervariable Regions ofrrsGenes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis
Top Cited Papers
Open Access
- 1 August 2004
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 70 (8) , 4800-4806
- https://doi.org/10.1128/aem.70.8.4800-4806.2004
Abstract
Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (Tm(L)) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to Tm(L). The average Tm(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.Keywords
This publication has 49 references indexed in Scilit:
- Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial CommunitiesApplied and Environmental Microbiology, 2003
- Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNAFEMS Microbiology Ecology, 2003
- Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to miceFEMS Microbiology Ecology, 2003
- Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacteria in the cecumFEMS Microbiology Ecology, 2002
- 16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel ElectrophoresisApplied and Environmental Microbiology, 2002
- Molecular Monitoring of Succession of Bacterial Communities in Human NeonatesApplied and Environmental Microbiology, 2002
- Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian SausagesApplied and Environmental Microbiology, 2001
- Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular TechniquesApplied and Environmental Microbiology, 2001
- Development of a Six-Stage Culture System for Simulating the Gastrointestinal Microbiota of Weaned InfantsMicrobial Ecology in Health & Disease, 2001
- Bioaugmentation with resin-acid-degrading bacteria enhances resin acid removal in sequencing batch reactors treating pulp mill effluentsWater Research, 2001