Biology Reference
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samples. Isolated cultures can be characterized by DGGE and can be assigned to fi eld populations
based on their DGGE profi les (Fig. 14). There are two advantages of this method. This method helps
to (i) assess community structure and (ii) provides relatively crude estimates of species diversity.
The disadvantages are that it involves comparative insensitive staining techniques and does not
provide information concerning specifi c phylogenetic groups that comprise a microbial community.
GC fractionation is shown to enhance bacterial community diversity assessment and the detection
of minority populations of bacteria is quite feasible by DGGE. Applying this technique, Holben
et al
. (2004) subjected bacterial community DNA (total bacterial community of cecum of broiler
chicken worldwide) to GC fractionation by differential density imposed by AT-dependent DNA
binding dye bis-benzimidazole. Such GC-fractionated DNA was PCR amplifi ed and subjected to
DGGE analysis. By this approach, it was possible to identify a number of phylotypes that were not
recovered using traditional random cloning and sequencing approach. Also directed cloning and
sequencing of individual bands from DGGE lanes corresponding to G+C fractions allowed detection
of numerous phylotypes that were not recovered by other traditional methods. Using PCR-DGGE,
total community structure of cyanobacteria has been determined in the mats inhabiting the intertidal
Figure 14:
Flow-chart showing different steps in PCR-DGGE.
