DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sediments
Open Access
- 24 August 2005
- journal article
- Published by Wiley in Environmental Microbiology
- Vol. 8 (2) , 308-320
- https://doi.org/10.1111/j.1462-2920.2005.00896.x
Abstract
In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H′) and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of β-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples.Keywords
This publication has 54 references indexed in Scilit:
- Maximum growth rates and possible life strategies of different bacterioplankton groups in relation to phosphorus availability in a freshwater reservoirEnvironmental Microbiology, 2006
- Richness and Diversity of Bacterioplankton Species along an Estuarine Gradient in Moreton Bay, AustraliaApplied and Environmental Microbiology, 2004
- Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availabilityEnvironmental Microbiology, 2004
- Viral effects on bacterial community composition in marine plankton microcosmsAquatic Microbial Ecology, 2004
- Covariance of bacterioplankton composition and environmental variables in a temperate delta systemAquatic Microbial Ecology, 2003
- Quantification, base composition, and fate of extracellular DNA in marine sedimentsLimnology and Oceanography, 2002
- Molecular characterization of estuarine bacterial communities that use high- and low-molecular weight fractions of dissolved organic carbonAquatic Microbial Ecology, 2001
- Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communitiesJournal of Microbiological Methods, 1998
- Improved soil dispersion procedures for total bacterial counts, extraction of indigenous bacteria and cell survivalJournal of Microbiological Methods, 1996
- Non‐parametric multivariate analyses of changes in community structureAustralian Journal of Ecology, 1993