MICROBIAL MONITORING DURING BIOAUGMENTATION WITH DEHALOCOCCOIDES - Bioaugmentation for Groundwater Remediation - page 187

6.7.3 Terminal-Restriction Fragment Length Polymorphism
Terminal-restriction fragment length polymorphism (T-RFLP) analyzes microbial
communities using PCR-amplified DNA for the gene of interest subjected to digestion with a
restriction enzyme (Liu et al., 1997 ). Restriction enzymes cut DNA at very specific basepair
sequences, typically 4-6 nucleotides in length. Since each amplified PCR product from a
different species is unique, the restriction enzymes cut the DNA at different points. For
example, one restriction enzyme might digest DNA from a species into two 600-basepair
fragments, while cutting DNA from a second species into a 300- and 900-basepair fragment.
Although functional genes are sometimes used for T-RFLP, its most common application is
on 16S rRNA genes. PCR-amplification occurs as described in the previous section; however,
one primer contains a fluorophore (much like the probes discussed in the earlier sections). After
PCR-amplification and digestion using a restriction enzyme, one terminal fragment of each
digested PCR amplicon contains the fluorophore, while the rest are unlabeled. The fragments
are separated by size using electrophoresis, with smaller fragments migrating faster. As the
terminal restriction fragments (TRFs) migrate past a sensor, those fragments with a fluoro-
phore are detected, with larger concentrations of fragments producing greater signal intensi-
ties. Those fragments lacking the fluorophore pass through undetected (hence, only terminal
fragments are measured). Through the proper selection of restriction enzymes, each TRF
represents one (or a small portion) of the microbial community members. Shifts in the
community composition result in varied numbers and signal intensities for the TRFs present,
permitting semi-quantitative interpretation between samples. In general, a greater number
of TRFs indicates greater community complexity. Determining the exact species associated
with a given peak requires prior sequence knowledge, usually obtained through cloning and
sequencing.
6.7.4 Denaturing Gel Gradient Electrophoresis
Denaturing gel gradient electrophoresis (DGGE) uses PCR-amplified target genes of
interest and a gel composed of a gradient of denaturing agents, commonly formamide and
urea (Madigan et al., 2003 ). The amplicon pool (all of approximately equal size) is separated by
electrophoresis based on the melting profiles unique to their nucleotide sequences. As the
double-stranded PCR-amplified DNA migrates through the gradient into increasingly higher
concentrations of denaturing agent, the amplicons melt (denature) and cease migration. While
the PCR amplicons are approximately the same size, the differences in melting properties result
from the varied nucleotide sequences located between the ends selected by the primers.
Visualization and comparison of the DNA bands after separation permits an evaluation of
the community complexity (Figure 6.8 ). A larger number of bands generally represents a more
complex and diverse microbial community. Once the fragments are separated, individual DNA
bands can be excised and sequenced to determine the species present within a community.
6.7.5 Temperature Gel Gradient Electrophoresis
Temperature gel gradient electrophoresis (TGGE) is very similar to DGGE, but instead of
using a gradient of denaturing agents, a temperature gradient is employed. As the DNA
fragments migrate through the gel and up a temperature gradient, they will denature and
further migration is retarded. Depending upon the nucleotide composition of the PCR ampli-
cons, they will denature at varying temperatures. As with DGGE, differences in band intensity