DNA ﬁngerprinting of microbial communities
The past two decades have seen a revolution in the application of molecular approaches to microbial ecology, offering the chance to study microbial communities and diversity via analysis of nucleic acids directly extracted from environmental samples (1). Moreover the application of a variety of DNA ﬁngerprinting techniques, mostly adopted from other ﬁelds of molecular biology, has allowed investigation of both variation in microbial communities, in particular via study of ribosomal RNA (see Chapter 2) and variation between individual functional genes. The application of DNA ﬁngerprinting has proved to be particularly valuable in the rapid generation of characteristic barcode-like patterns for entire microbial communities derived from large numbers of environmental samples. Fingerprinting is useful for readily monitoring changes in microbial communities in time and space with minimal effort, which is in stark contrast to the often astronomic diversity of microbial communities in natural habitats. The ﬁngerprint is a drastically simpliﬁed representation of the microbial community, deﬁned ﬁrstly by the speciﬁcity of the oligonucleotide primers used to generate the PCR amplicons of interest and secondly by the preferential ampliﬁcation of DNA fragments during PCR. Separating complex PCR products into ﬁngerprints yields single bands for predominant sequences but rare sequences may often remain undetected. However, in very complex environmental communities the few predominant species may well constitute only a minority while thousands of rare species contribute most of the biomass (and hence DNA) but no signal in the ﬁngerprint. This loss of information should be considered as a drawback of any PCR-based investigation of environmental samples. By contrast, the application of ﬁngerprinting avoids some of the other pitfalls of PCR (2) such as the formation of chimeric DNA amplicons during PCR (3; See also Chapter 2, Section 2.2.2). Such artiﬁcially created molecules when cloned can lead to problems in subsequent phylogenetic analysis of clone libraries (4).