title: Improvements of polymerase chain reaction and capillary electrophoresis single-strand conformation polymorphism methods in microbial ecology: toward a high-throughput method for microbial diversity studies in soil.
abstract: The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.
authors.affiliation: Laboratoire d'Ecologie Alpine, Perturbations Environnementales et Xénobiotiques, CNRS UMR 5553, Université Joseph Fourier, Grenoble Cedex 9, France.
authors.forename: Lucie
authors.initials: L
authors.lastname: Zinger
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gury
authors.forename: Frédéric
authors.initials: F
authors.lastname: Giraud
authors.forename: Serge
authors.initials: S
authors.lastname: Krivobok
authors.forename: Ludovic
authors.initials: L
authors.lastname: Gielly
authors.forename: Pierre
authors.initials: P
authors.lastname: Taberlet
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Geremia
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics*; D044822:Biodiversity; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D017753:Ecosystem; D019075:Electrophoresis, Capillary / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase