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

significantly reduced the likelihood of erroneous insertions during the extension phase of
PCR. However, there were still bands within the profiles which could not be explained.
These bands were usually very faint and so were not designated for analysis by the
automated band selection software. The majority of artefactual bands on a DGGE gel
especially high up in the gel (closer to the wells) are often single stranded DNA due to
incomplete extension during PCR amplification (Muyzer & Smalla, 1998). The final
extension time for the DGGE-PCR in the current study was 10 minutes which was
considered long enough to ensure complete extension of all PCR products (Ercolini c't al.,
2001 a). Incomplete extension of the DNA strand during PCR can be caused by the GC-
clamp, which leads to multiple bands from a single product (Nübel et al., 1996). The
confirmation of the absence of incomplete strand synthesis by attempted re-amplification
of bands within the DGGE profiles (incomplete strands would not re-amplify) was not
determined in the current study due to time constraints which prevented assessment of
each band within the community profiles.
6.3.2.4.4. -
PCR contamination
Fourthly, the possibility of contaminating DNA being amplified within the PCR
which would cause a complete misrepresentation of the community diversity. The
sensitivity of PCR required strict aseptic technique to prevent the amplification of DNA
from bacterial contaminants (van Wintzingerode et al., 1997). However, bacterial
contaminants in the current study cannot be ruled out as time constraints prevented full
analysis of the DNA bands present within the DGGE profiles. Characterisation of PCR
contaminants in DGGE profiling can be achieved by sequence identification of the bands
within a profile. Identification of a common PCR contaminant (e. g. Bacillus sp. ) would
mean re-amplification
of the DGGE profiles. To prove a putative contaminant
conclusively would require the use of techniques such as FISH, which utilise labelled
species specific DNA probes applied in situ to confirm or deny the presence of an isolate
in a community (Dang & Lovell, 2002). The diversity of the DGGE community profiles
obtained during the current study and the distinct division of community profiles betx\ecn
brackish and hypersaline lakes (section 6.2.2) infers that the community profiles do not
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