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The reaction mix contains both deoxynucleotides (A, T, C& G) and dideoxynucleotides
at concentrations which produce a finite probability that a dideoxynucleotide will be
incorporated into each nucleotide position in a growing chain. The incorporation of a
dideoxynucleotide inhibits further elongation, hence chain termination. This produces a
number of truncated chains of varying length. This can then be read in four different
reactions (ddATP, ddCTP, ddTTP, and ddGTP) to produce the order of nucleotides in the
original template. Alternatively,
a nucleotide-specific label can be attached to each
dideoxynucleotides and the reaction read off on a polyacrylamide gel (QIAGEN, 1995).
The template used is generally the 16s rRNA gene. This gene is involved in the
structure of ribosomes and the reading of mRNA for the production of proteins. There are
three rRNAs used for taxonomic identification of bacteria; the 5S, 16S and 23S
molecules. They are used to indicate relatedness in bacteria because they are present in
all bacterial species. They have extensive regions of conserved sequence allowing
relatedness to be assessed between species which otherwise share no sequence similarity,
showing enough variation to allow differentiation of even closely related species. This
allows phylogenetic relationships (suggested evolutionary relationships) to be inferred
from the variations of these genes (Priest and Austin, 1995). The 16S rRNA molecule is
generally accepted as the molecule upon which most of modern bacterial taxonomy is
based (Priest & Austin, 1995; Palys et al., 1997; Dalevi et al., 2001). This is because the
5S molecule is too small (116-120bp) as it undergoes marked mutational changes which
would be obscured by long sections of conserved sequence in the 16S rRNA, and the 23 S
molecule is too large to be easily sequenced (Priest and Austin, 1995). Over the last
seven years, since the publication of the first bacterial genome sequence, over 60
bacterial genome sequences have been published and there are more than 200 current
research projects concerning bacterial genome sequencing (Suerbaum et al., in press).
However, the 16S rRNA gene is still used as the primary identification tool for culture-
dependant and -independent
bacterial characterisation, this is exemplified by the
presence of more than 30,000 16S rDNA sequences available in the main ribosomal
databases, GenBank, EMBL and DDBJ (Dalevi et al., 2001). When using the 16s rDNA
gene, the amplified product can either be directly sequenced (Bevan et al., 1992; Kocher,
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