Biomedical Engineering Reference
In-Depth Information
6.7 COMMUNITY PROFILING
The advent of molecular techniques, PCR in particular, provided the first opportunity for
the broad investigation of environmental microbial communities. Links between the community
population structure and metabolic functions in native environments are being determined as a
result of many of the techniques described below. The majority of the technologies described
below require PCR amplification of environmental DNA, making them subject to the PCR
biases and limitations described in an earlier section. The analysis of PCR amplicons (the pool
of amplified target gene DNA) provides relative information only for the timepoint at which
the environmental sample was taken. The true power of these techniques comes not from a
single snapshot, but through comparing multiple timepoints and determining changes in
community structure resulting from varying environmental conditions (e.g., diurnal patterns,
saline/freshwater interfaces, aerobic slugs of groundwater, etc.).
6.7.1 Gel Electrophoresis
Conventional gel electrophoresis, as a stand-alone technique, does not result in
community profile data. However, it is a commonly used DNA separation technique that is
often used in conjunction with other DNA separation and profiling methods, and thus a brief
overview is warranted. Gel electrophoresis relies on the differential migration of DNA with
different sizes or secondary structures through a semi-solid, but porous, gel matrix. DNA,
being negatively charged, will migrate along a gel toward the positive electrode if subjected
to an electric field. In gels of a uniform nature, the larger a fragment of DNA, the more the
gel retards movement and the shorter the distance that fragment travels in a given time.
Similarly, in gradient gels, DNA becomes denatured and occupies more physical space,
subsequently slowing movement. Thus, much like chromatography permits separation
of mixed gases or chemicals in solution, electrophoresis separates mixed fragments of DNA.
The DNA is visualized using a variety of staining techniques, with individual blocks of
DNA referred to as “bands.” The intensity of these bands roughly corresponds with the
concentration of DNA.
6.7.2 Cloning and Sequencing
Cloning and sequencing of target genes of interest offer the most informative (although
not necessarily the most economical) method of determining microbial community composi-
tion. This technique uses PCR-amplified target genes of interest, with both group-specific genes
(e.g., 16S rRNA) and functional genes (e.g., vcrA ) used with great frequency. Individual PCR
amplicons from the pooled mixture are ligated into plasmids (small circular pieces of extra-
chromosomal DNA), which in turn are inserted into Escherichia coli host cells. This process,
termed “transformation”, results in each E. coli host cell containing a single plasmid with a
single PCR amplicon. Thus, when cells are grown on media plates, each colony (derived from a
single E. coli cell) contains a DNA fragment representative of a single target gene. Extracted
DNA from these colonies is analyzed using sequencing technologies, producing hundreds
(or thousands) of DNA sequences from the initial PCR amplicon pool. Comparison of the
generated sequences with molecular databases provides information on the identity of the
organism from which the target gene was derived. Phylogenetic and ecological comparisons
are commonly used for determining the complexity of the microbial communities sampled,
as well as for inferring some degree of metabolic functionality.
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