Flow diagram showing the different steps in the analysis of microbial
community structure by PCR-DGGE. DNA is extracted from an
environmental sample and used as template in the polymerase chain
reaction (PCR) to amplify the 16s rRNA encoding genes of bacteria.
Thereafter, the PCR products are separated by denaturing gradient gel
electrophoresis (DGGE) (lane 1). The phylogenetic affiliation of the
predominant community members, as represented in the DGGE band
pattern, can be inferred by comparative analysis of sequences from
excised and re-amplified DNA fragments (lanes 2-7) and sequences
stored in nucleotide databases. Moreover, the sequence information
can be used to design an oligonucleotide probe for the detection of a
specific bacterial population by fluorescence in situ hybridisation
(FISH). (a) A photograph of a microbial community stained with the
DNA-binding fluorochrome DAPI resulting in blue light emission from
all bacteria after UV illumination. (b) The same microbial community
after incubation with a red fluorochrome-labelled oligonucleotide probe
specific for the bacterial population represented by the sequence
obtained from band 3 in the denaturing gradient gel.