Microbial Diversity: Its Exploration and Need of Conservation - Applied Environmental Biotechnology: Present Scenario and Future Trends - page 51

Fig. 4.3 RFLP helps us see the differences between the DNA

Then the comparison of the ribosomal DNA
sequences with known rRNA sequences was
done. (Muyzer and Smalla 1998 ; Nakatsu et al.
2000 ) Although this technique does not give the
full description but numbers and lineages of mi-
croorganisms within environmental samples can
be determined, remarkably their phylogenetic re-
lationships and genetic similarity to sequences in
known databases.
Many scientists have amplified 16S rRNA
genes from environmental samples, and have
produced important results from such works
(Giovannoni et al. 1990 ; Fuhrman et al. 1993a ;
Mishra and Thakur 2010 ). This is still a time-
consuming technique even after simplification of
steps and is not suitable for analysis of hundreds
of samples and along with requires a relatively
large database (Colwell and Hawksworth 1991 ).
It is now possible to study microbial commu-
nities using the randomly-amplified polymor-
phic DNA (RAPD) method. This generates DNA
fingerprinting characteristics of the community.
This technique also does not allow the identifica-
tion of individual microorganism.
Restriction fragment length polymorphism
(RFLP) is another tool used to study microbial diver-
sity and community structure (Moyer et al. 1996 ).

This method relies on DNA polymorphisms. In
this method, electrophoresed DNA bands are
blotted from agarose gels onto nitrocellulose or
nylon membranes and hybridized with appropri-
ate probes prepared from cloned DNA segments
of related organisms (Fig. 4.3 ). RFLP has been
found to be very useful particularly in combina-
tion with DNA-DNA hybridization and enzyme
electrophoresis for the differentiation of closely
related strains (Rastogi and Sani 2011 ). RFLPs
may provide a simple and powerful tool for the
identification of bacterial strains at and below
species level (Kauppinen et al. 1994 ). This meth-
od is useful for detecting structural changes in
microbial communities. But this technique can-
not be used as an extent of diversity or for de-
tection of specific phylogenetic groups (Liu et al.
1997 ). Banding patterns in diverse communities
become too complex to analyze using RFLP since
a single species could have four to six restriction
fragments (Tiedje et al. 1999 ).
Terminal restriction fragment length poly-
morphism (T-RFLP) is a technique that talks
about some of the limitations of RFLP (Zhang
et al. 2008 ). This technique is an extension of
the RFLP/amplified ribosomal DNA restriction