Biology Reference
In-Depth Information
A controversy seems to have arisen on the suitability of fi ngerprinting methods for the community
studies. While community fi ngerprinting methods such as automated ribosomal intergenic spacer
analysis (ARISA) and T-RFLP are useful for comparative analyses, they are not useful to assess the
phylotype richness or community evenness because the values for these have been found to be
variable and appear to be dependent on restriction enzyme used for generating the profi le. Although
T-RFLP provides a rapid means for comparing the relationships between bacterial communities,
it may not be suitable for highly complex communities (Dunbar
et al
., 2000). Subsequently, these
workers assessed phylogenetic relationship of every group of 16S rDNA sequences that yielded the
TRFs of the same size. When replicate samples (aliquots of a single restriction digest) were compared
the profi les substantially differed from one another suggesting that the results are not reproducible.
However, phylogenetic inferences will be most effective if TRF profi les represent only a single
bacterial division or a smaller group. The analytical precision of this method is doubtful as about 85%
of the 169 TRF profi les were not reproducible when nine replicate samples from a single soil sample
were analysed. Such methods are inherently limited by their detection threshold or, more precisely,
by their dynamic range (Dunbar
et al
., 2001). Danovaro
et al
. (2006) compared microbial community
diversity and richness estimates obtained by using ARISA with T-RFLP of 16S rRNA genes. They
showed that ARISA estimates of bacterial species richness are always higher than those obtained
using T-RFLP analysis. Bent
et al
. (2007) argued that the number of peaks detected in either T-RFLP
or ARISA assays grossly underestimate the actual richness of any community. According to them, the
two methods cannot be treated at par, as the resolving power of each of these is different and thus
can partition the community differently. Moreover, the observed categories do not correspond to the
named taxonomic identities. They concluded that the “current microbial community fi ngerprinting
methods cannot provide reliable diversity indices” and also added that it is not understandable
why the investigators do not realize the limitations of such methods and hastened to add that “the
emperor has no clothes”. Danavaro
et al
. (2007), however, observed that T-RFLP analysis and ARISA
in fact lead us to clearly distinguishable taxonomic categories. It is now well realized that the 16S
rRNA gene alone is not suffi cient to defi ne phylogenetic relationships among closely related species
(Normand
et al
., 1996; Guasp
et al
., 2000; García-Martinez
et al
., 2001; Song
et al
., 2004; Brown
et al
.,
2005). In view of this, 16S rRNA-23S rRNA ITS region emerged as an important molecular marker
for discrimination to the species level and even within species (Guasp
et al
., 2000; Goncalves
et al
.,
2002; Xu and Cote, 2003; Song
et al
., 2004; Kwon
et al
., 2005). The criticism of Bent
et al
. (2007) to
ARISA and T-RFLP can also be extended to any fi ngerprinting technique including DGGE/thermal
gradient gel electrophoresis (TGGE). They concluded that all fi ngerprinting techniques are always
useful in comparing biodiversity of different habitats and ecosystems. Fingerprinting techniques are
akin to prět-a-porter clothes. Although such a dress is not as glamorous as the dress of a top stylist
but the fact remains that at least “it does not leave you naked”.
vi)
PCR and DGGE:
The sixth method is dependent on DNA melting behavior. In DGGE, chemicals
such as urea or formamide gradients are used to separate the PCR products. This has been developed
by Muyzer
et al
. (1993, 1995) in which cellular/environmental DNA sample is subjected to PCR
amplifi cation by universal or specifi c domain primers to identify 16S rRNA gene or any other target
gene. The amplifi ed gene products are then subjected to DGGE, stained and visualized for profi le
and data analysis. The total number of bands represents the total community structure and each
band is represented by the individual species/strain component and the intensity of the band in turn
refl ects the abundance of particular species/strain. This technique involves sequence-dependent
separation of PCR products which can be used to assess the genotypic diversity in environmental
