Agriculture Reference
In-Depth Information
6.3.4.4
Restriction Fragment Length Polymorphism (RFLP)
This method relies on the DNA polymorphism and has been applied to estimate the
diversity and community structure in different environments (Moyer et al. 1996 ).
This method, in combination with DNA hybridization and electrophoresis, provides
a powerful tool for strain typing and also for the determination of intra-species
variation. The method has been successfully used for the detection of specific
phylogenetic groups within a community; however the complexity of the band-
ing patterns limits the application of this method for analysing bacterial diversity
(Tiedje et al. 1999 ).
6.3.4.5
Terminal Restriction Fragment Length Polymorphism (T-RFLP)
T-RFLP is an extension of RFLP/ARDRA and addresses their shortcomings; it pro-
vides an easy and powerful tool for microbial community analysis in various envi-
ronments. In this method, one set of the PCR primer is labelled with a fluorescent
dye, such as TET (tetracholoro-6-carboxyfluoroscein) or 6-FAM (phosphoramidite
fluorochrome 5-carboxyfluorescein). The generated Amplicons are digested with
restriction enzymes and electrophoresed using an agarose gel. The labelled frag-
ments termed as T-RF's are separated and analysed using an automated sequencer.
Each unique fragment length is described as an Operational Taxonomic Unit (OTU)
and the frequency of each OTU can be counted and used as a measure of diversity
and richness of the microbial community (Liu et al. 1997 ). This method has been
applied to determine bacterial diversity and community succession during compost-
ing in different environments. T-RFLP has been considered a highly effective tool
for comparing the relationships between different environmental samples and also
in the measurement of spatial and temporal changes in bacterial communities with
five times greater success rate than DGGE (Tiedje et al. 1999 ).
6.3.4.6
Denaturant Gradient Gel Electrophoresis (DGGE)/Temperature
Gradient Gel Electrophoresis (TGGE)
DGGE or TGGE allows the separation of DNA fragments with the same length but
with different base pair sequences. The method involves the amplification of the 16S
or 18S rRNA gene and separation of the partially melted DNA molecules in acryl-
amide gels containing a linear gradient of DNA denaturants (urea and formamide).
The variation in the nucleotide sequences within the DNA generates different frag-
ments based on the differences in melting points. DNA sequences with a difference
of only one base pair can be separated using DGGE. TGGE is based on the same
principle as DGGE, but in this method a temperature gradient is employed in place of
chemical denaturants. The method is cost-effective, reproducible, reliable and rapid
(Muyzer 1999 ). DGGE has been used to monitor microbial community profiles and
succession in a variety of composts (Ishii et al. 2000 ; Mühling et al. 2008 ).
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