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hybrids should be equal. However, Suzuki and Giovannoni (1996) have shown that bias
can occur due to successive reannealing of a single template, which would progressively
inhibit the formation of other primer-template hybrids. Van Wintzingerode et al. (1997)
have suggested that this should not occur if the community genomic DNA template
contains sufficient diversity. Hansen et al. (1998) suggested that DNA bias during PCR
originated in the DNA which flanked the PCR amplicons, i. e. some species may have
DNA inserts prior to the 16S rDNA which inhibits the amplification of the target DNA.
For the current project it is unknown if this bias has affected the results. It is possible that
preferential amplification of the gene products of specific species within the community
acted to inhibit the amplification of other species, which could explain the absence AFP
active cultured isolates from the community profiles. Bosshard et al. (1999) have
suggested that only numerically dominant groups will be amplified in community PCR.
Ercolini et al. (2001b) have suggested that DGGE using the variable V3 region is prone
to selective amplification, they suggested using culture dependant analysis to support
culture-independent analysis due to the bias present in both analyses. This has been
previously substantiated by other research (Liesack et al., 1997). During the current study
nested PCR was used, which incorporates two PCR steps (Section 2.9.5.3). This was
performed as V3 PCR amplification from genomic DNA was not providing repeatable
results, possibly due to contamination in the genomic DNA preparation. The inclusion of
multiple PCR steps in the current study would have increased the likelihood of bias. The
probability of differential amplification could also suggest why, for example.
isolate 302
was represented in the community profile for Ace Lake, 4m from September despite not
being cultured from this community. It may not have been cultivatable from the
environmental conditions in Ace Lake, but was preferentially amplified by the multiple
DGGE-PCRs.
6.3.2.4.3 -
Artefactual PCR products
Thirdly the production of artefactual PCR products such as chimeric molecules
and erroneous base pair insertions during the extension phase can affect a DGGE profile
by altering the composition of the target sequence. During the current study a 'proof
reading' enzyme was associated with the Taq polymerase which should have
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