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compared the effects of three different DNA extraction methods (lysozyme digestion, CTAB
method and bead beating) on the analysis of different micro-ecological environments in a
grass carp (
Ctenopharyngodon idella
) farm pond (pond sludge, feed) and the gut microbiota
by 16S rRNA V3 DGGE. The results revealed differences in the DGGE fingerprints from the
different DNA extraction methods. It is important therefore that the DNA extraction meth-
ods used should be robust and optimized before the analysis of GI microbial communities.
Additional problems associated with PCR based techniques include heteroduplex detection,
detection of chimeric rRNA operons, and low sensitivity to rare members present in mixed
microbial communities (McCartney 2002).
5.2.2 Electrophoresis based methods
Although RISA (Navarrete
et al
. 2009), length heterogeneity PCR (Heikkinen
et al
. 2006),
terminal restriction fragment length polymorphism (T-RFLP) (Moran
et al
. 2005; Fjellheim
et al.
2012) and temporal temperature gradient electrophoresis (TTGE) (Navarrete
et al
. 2009;
2010; 2012) have been utilized to characterize the gut microbiota of fish, DGGE remains the
most commonly utilized electrophoresis based method due to its relatively inexpensive and
simple nature. DGGE, and TTGE, are based on the electrophoresis of targeted regions of the
16S RNA gene which display differing denaturing properties depending on the nucleotide
composition; typically the 16S rRNA V3 region (after Muyzer
et al
. 1993) is used but the
V6-V8 (Zhou
etal
. 2009a; Desai
etal
. 2012) and V4 (Bjornsdottir
etal
. 2009; Hermannsdottir
etal
. 2009) regions have also proven to be phylogenetically informative. RISA analysis targets
the electrophoresis of the ribosomal intergenic spacer region between the small (16S) and large
(23S) ribosomal subunits, which varies in length (from ca. 150 to 1500 nucleotides (nt)) in
different bacterial strains. This approach may allow for a higher degree of identification in
subsequent sequencing given the possibility of sequencing longer PCR amplicons.
These electrophoresis methods can provide a fast, inexpensive method for assessing the
dominant gut microbiota of fish and the effects of prebiotics and probiotics on these popu-
lations. Fingerprinting profiles based on 16S rRNA are reported to represent 90-99% of the
bacterial community (Zoetendal
et al
. 1998). A range of subsequent statistical methods (based
on either binary or weighted analysis of banding patterns) have been developed to compare
fingerprints. Such methods allow comparisons of profiles from samples to be made by using
similarity matrices, dendrograms, and non-metric multidimensional scaling (nMDS) which
show the relatedness of the microbial profiles, and principal component analysis (PCA) can
be used to determine the influence of environment, host, or other factors on the phylogenetic
patterns. Ecological statistics, such as species richness, species diversity and one-way analysis
of similarity (ANOSIM) can also be applied. In order to gain information on specific bacteria,
sequencing of excised bands is essential. However, given the low number of nucleotides used
(e.g. ca. 150 nt for the V3 region of the 16S rRNA gene) a high level identification, and the
ability to resolve between highly similar strains, are often restricted. As such, identification
beyond genus level can be difficult. Another issue is the potential for PCR amplicons with
similar or identical denaturing characteristics to migrate to the same location in the lane, thus
providing difficulty in gel interpretation and subsequent sequencing of operational taxonomic
units (OTUs). Particular care should be taken when comparing samples from different gels, as
the normalization of bands across gels can introduce artifactual diversity. Even when using a
set of markers in each gel, it is advisable to conduct within gel comparisons of key treatment
groups. Experimental design should take this factor into account.
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