Molecular approaches to the assessment of biodiversity in aquatic microbial communities

For the past 20 years, the increased development and routine application of molecular-based techniques has made it possible to carry out detailed evaluations of the biodiversity of aquatic microbial communities. It also offers great opportunities for finding out how this parameter responds to various environmental stresses. Most of these approaches involve an initial PCR amplification of a target, which is generally located within the ribosomal operon. The amplification is achieved by means of primers that are specific to the organisms of interest. The second step involves detecting sequence variations in the PCR fragments either by a cloning/sequencing analysis, which provides a complete characterization of the fragments, or by an electrophoretic analysis, which provides a visual separation of the mixture of fragments according to sequence polymorphism (denaturing or temperature gradient gel electrophoresis, single strand conformation polymorphism) or length polymorphism (terminal-restriction fragment length polymorphism, automated ribosomal intergenic spacer analysis). Other non-PCR-based methods are also commonly used, such as fluorescence in-situ hybridization and DNA re-association analysis. Depending on the technique used, the information gained can be quite different. Moreover, some of these analyses may be rather onerous in terms of time and money, and so not always suitable for screening large numbers of samples. The most widely used techniques are discussed in this paper to illustrate the principles, advantages and shortcomings of each of them. Finally, we will conclude by evaluating the techniques and discussing some emerging molecular techniques, such as real-time PCR and the microarray technique.