Extraction of Prokaryotic Genomic DNA from Marine Microbial Communities Suitable for Amplification Using the Polymerase Chain Reaction

Abstract

AbstractWe present a method for extraction of DNA from marine microbial communities and amplification of the small subunit ribosomal RNA gene from the prokaryotic organisms contained therein. Results of cloning, sequencing and phylogenetic analyses are also presented. This DNA extraction technique is specifically designed to be used in conjunction with the Polymerase Chain Reaction (PCR). Emphasis was placed on producing a technique which results in total cell lysis yielding DNA of sufficient purity to facilitate amplification using PCR. Minimisation of surface contamination and procedure suitability for a multi‐sample study were considered to be of prime importance throughout this investigation. As a demonstration of the suitability of the technique, a preliminary analysis of the small subunit ribosomal RNA (SSU rRNA) gene from marine microbial communities was investigated. The amplification reactions are designed to specifically target the SSU rRNA gene from the two primary prokaryotic domains: Bacteria and Archaea sensu Woese (Woese et al., 1990). The primers were designed to amplify a sequence exceeding 1 Kb in length which is sufficient for use in taxonomic and phylogenetic analyses of marine communities (Murray and Schleifer, 1994). The samples used represent spatially and temporally distinct regimes, from the North East Atlantic. These include water samples from open ocean sites and gut contents from a deep sea deposit feeder, Oneirophanta mutabilis (Holothuria: Elasipodida). The analysis shows the identification of a group of unusual Archaea. as‐yet uncultured, in the samples analysed.