ABSTRACT
Over 20 years ago, the first protocol titled ‘DNA extraction from soil’ was published by Torsvik (1). However, only in the late 1980s/early 1990s, when molecular tools such as nucleic acid hybridization, the polymerase chain reaction (PCR) and DNA cloning and sequencing became increasingly available, more attention was focused on the analysis of DNA extracted from environmental bacteria without prior cultivation. Obviously, the analysis of nucleic acids extracted directly from environmental samples allows the researcher to investigate microbial communities by obviating the limitations of cultivation techniques. The phenomenon that only a small proportion of bacteria can form colonies when traditional plating techniques are used (2) was first described by Staley and Konopka (3) as the great plate anomaly. A further limitation of the cultivation-based studies of microbial communities is that under environmental stress bacteria can enter a state termed ‘viable but non-culturable’ (vbnc), and again these bacteria would not be accessible to traditional cultivation techniques (4, 5). Consequently researchers were attracted by the opportunities afforded by analyzing nucleic acids recovered directly from environmental samples that should be representative of the microbial genomes present in such samples. The analysis of DNA can provide information on the structural diversity of environmental samples, or on the presence or absence of certain functional genes (e.g. genes conferring xenobiotic biodegradative capabilities, antibiotic resistance or plasmid-borne sequences), or to monitor the fate of bacteria (including genetically modified organisms) released into an environment. However, in general the analysis of DNA does not allow conclusions to be drawn on the metabolic activity of members of the bacterial or fungal community or on gene expression. This information might be obtained from analysis of RNA (rRNA or mRNA) (see also Chapter 5).
