Molecular microbial community analysis: Understanding and overcoming the limitations

The field of microbial ecology has seen great advances in the past decade, owing in part to the development of molecular methods for microbial community analysis. The advent of molecular tools has allowed for the exploration of the rich microbial diversity of the biosphere without the need to first culture the organisms of interest. Although ecological studies vary in scope from the identification of new and intriguing microorganisms to the complex interactions of mixed microbial communities, the ability to infer ecological significance depends on confidence in the molecular techniques used. Techniques should be accurate and sensitive with an ability to finely discriminate between different kinds of organisms. The techniques should also be broad in their coverage and should represent the true abundance of the different microbial populations. Current technology based on the 16S ribosomal RNA gene and the Polymerase Chain Reaction may address one or two of these aspects, but no technique exists that can adequately address them all.; This body of research describes a novel approach to exploring microbial diversity using paramagnetic bead-based gene capture probes and a random PCR amplification protocol. This new technique, termed "CAPRA," overcomes the constraints of traditional 2-primer PCR in terms of primer design and primer-based amplification bias, and allows for the study of protein-encoding genes. A set of oligonucleotide capture probes was generated to target a strictly conserved region of the rpoC gene, which encodes for a subunit of the DNA-dependent RNA polymerase. Cloning of captured and randomly amplified material from a pure culture suggested a signal to noise ratio of approximately 1:6, representing an enrichment of the rpoC gene by 1250 times. Further experiments with pure cultures, model communities, and unknown environmental samples suggest that CAPRA-based analyses are sensitive, accurate, and quantitative. This combination offers a powerful tool for the study of microbial communities and the future of molecular microbial ecology.