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

All PCR-based rRNA molecular techniques have substantial bias (Amann et al.,
efficiently (Amann et al., 1995). This is one reason for the culture bias seen in
environmental sampling, it is also another reason why traditional culturing techniques
should always be used in conjunction with molecular techniques (Hugenholtz et al..
1998).
6.3.2 -
DG GE bias
1995). This is exemplified by the current DGGE study. As stated, 9 AFP active isolates
were not present within the community profiles of the lakes from which they were
isolated, which suggests that the DGGE analysis does not represent the full diversity of
the community. As has already been suggested it is possible that, because the community
profiles selected for analysis were not directly sampled from the communities from which
the AFP active isolates were cultured, the AFP active isolates may in fact not be present
within those communities. However, the ubiquitous presence of Marinomonas protect
within Ace Lake during November (as indicated by its isolation in Ace Lake at 8m, 4m
and 2m during November (Table 2- Chapter 5)) suggests it is unusual that this species
should not be identified in the Ace Lake community profiles from November.
DGGE as with all molecular rRNA analyses has bias at virtually every physical,
chemical and biological step, all of which can cause artefacts and distortion of the
observed community structure (van Wintzingerode et al., 1997; Ercolini et al., 2001 b).
This can be seen in the current study, where the cultivated record does not correlate with
the molecular `view' of the community. There are five major steps in all small subunit
ribosomal molecular analysis and all involve some level of bias; the following sections
outline the areas of bias in the context of the current study.
6.3.2.1 -
Species composition of community
The selection of a molecular technique is largely dependant on knowing what
it is
that the technique will be selecting for, e. g. Archaea or Eubacteria. This is the first step
where bias can occur. It is important to design a protocol which will select for the species
present in that community. This can be determined by analysis of the environment, for
example. observation of the different morphotypes present within the community bý
fluorescent microscopy and physical and chemical analysis of the environment. which
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