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

(Neefs et al., 1990), amplified by PCR from community genomic preparations. The DNA
is separated through the mobility of partially melted DNA during electrophoresis in a
polyacrylamide gel, which will cease migration at specific locations along a denaturing
gradient (Muyzer et al., 1993; Muyzer & Smalla, 1998). DGGE can detect up to 95% of
all possible single base substitutions in sequences up to 1000bp (Myers et al., 1985). As
stated this has been applied to a wide variety of microbial populations including
meromictic lacustrine systems (f vreas et al., 1997; Bosshard et al.,
1999). hypersaline
pools (Kübel et al., 2000), Antarctic coastal waters (Murray et al., 1998). hot springs
(Ferris et al., 1996), varied marine systems (Muyzer et al., 1995; Schäfer et al., 2001;
Campbell & Cary, 2001), food products (Cocolin et al., 2001; Coppola et al., 2001;
Ercolini et al. 2001a & 2001b) and agriculture (Ampe & Miambi. 2000; Ampe el al.,
2001; Duineveld et al., 2001). DGGE analysis has also been used to study the diversity of
specific species groups (e. g. ammonia-oxidizing bacteria, Nicolaisen & Ramsing, 2002).
analyse sequence variation between type strains of the human immunodeficiency virus
(Andersson et al., 1993) and for the identification of specific genes of interest from
natural ecosystems (Muyzer, 1999).
In the current study, community samples were taken at 2m intervals from three of
the detailed study lakes (Ace Lake, Pendant Lake and Triple Lake, Chapter 3) and the
surface water of Deep Lake and Club Lake, at two time intervals between the late austral
winter and early austral summer. This was to determine the presence and nature of spatial
and temporal fluctuation within the microbial population and to observe how the AFP
active bacterial strains were represented in the community.
6.2 -
Results
DGGE analysis was performed using the method outlined in Muyzer et al.,
(1993; Section 2.9.5.4). Community and pure culture chromosomal DNA was extracted
using the CTAB protocol (Ausubel et al., 1999). Nested PCR of the V3 region
for DGGE
analysis was performed using a protocol outlined in Ercolini et al. (2001 a), from a1 kb
16S rDNA fragment (PCR protocol outlined in section 2.9.5.3).
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