ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

of bacterial isolates to the species, subspecies and strain level (Rademaker & de Bruijn.
1997).
1.6.3.8 -
DGGE and TGGE
The differentiation of multiple species from an environmental sample can be
performed by analysing base pair variations within a specific gene common to all species.
Microbial community diversity can be analysed by PCR amplification of the variable V3
region of the 16S rRNA gene (Neefs et al., 1990) from the total community genomic
DNA. This provides numerous copies of the V3 regions from every bacterial species in
the community (unless selective amplification occurs), which are then separated on a gel
by denaturing or temperature gradient gel electrophoresis (DGGE or TGGE) (Muyzer et
al., 1993; Muyzer & Smalla, 1998). The resulting fingerprint indicates the major species
within a given ecosystem and can be used to show temporal changes in microbial
community structure and dominance (Ovreäs et al., 1997; Murray et al., 1998; Muyzer &
Smalla, 1998; Muyzer, 1999; Ampe & Miambe, 2000; Bosshard et al., 2000; Ampe et al.,
2001; Cocolin et al., 2001; Coppola et al., 2001; Ercolini et al., 2001 a; Watanabe et al.,
2001).
1.6.4 -
Numerical taxonomy
In order to classify living organisms, it is necessary to compare them. Molecular
techniques can provide information on each organism which can then be compared with
all other organisms, thus providing a dataset which can demonstrate the relatedness of a
group of organisms. This is the basic principle behind numerical taxonomy. However,
there is no single correct way of characterising organisms (Priest & Austin, 1995). In fact
there are three distinct ways in which bacteriologists classify bacteria:
1. Special-purpose classifications, in which bacteria are classified based on
characteristics which are ideal for their classification within a particular
discipline. These are of no value to general microbiological classification (Priest
& Austin, 1995).
2. Phenetic classifications, which refers to relationships between organisms
(Operational Taxonomic Units, OTUs) based on comparison of the individual
characteristics of the complete organism, both its phenotype and genotype.
Placement of an organism within a group or `phenon' is based on the organisms
26