ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

1992), or cloned into another bacterium (e. g. E. coli) via a plasmid, and then sequenced
directly from the plasmid (Brown, 1996).
1.6.3.2 -
Amplified ribosomal DNA restriction analysis (ARDRA)
ARDRA uses the sub-species specific variations in the highly conserved 16S
ribosomal RNA gene to delineate phenetic relationships between bacteria (Vaneechoutte
et al., 1995; Vila et al., 1996; Ingianni et al., 1997; Jawad et al., 1998; Vinuesa et al.,
1998; Versalovic et al., 1998). The 16S rDNA fragment is amplified from genomic DNA
using PCR. The fragment is then digested using endonucleases to produce a sub-species
specific DNA profile (Rademaker & de Bruijn, 1997). ARDRA was chosen based on its
simplicity and flexibility
because a large number of unknown specimens needed to be
typed. ARDRA is an ideal tool for typing novel bacterial species (Ingianni et al., 1997)
and can be of help in elucidating the ecological significance of specific bacteria
(Vaneechoutte et al., 1993).
1.6.3.3 -
Southern hybridisation (ribotyping)
Genomic DNA is digested using endonucleases and then run on an
electrophoresis gel which is transferred (blotted) onto a nylon membrane. The DNA on
the `blot' is then hybridised with labelled 16S / 23S rDNA (32P or digoxigenin). The
labelled DNA is then detected providing a unique fingerprint pattern for individual
species or even strains. The technique was pioneered by Grimont and Grimont (1986).
Whereas other randomly primed or restriction enzyme methods of total bacterial DNA
(RFLP, rep-PCR, AP-PCR, etc. ) produce numerous bands which all have to be resolved
to enable sufficient species differentiation to be assessed, ribotyping produces only a
small number of distinct bands allowing discrimination of individual species and sub-
species (Grimont & Grimont, 1986).
1.6.3.4 -
Restriction fragment length polymorphism (RFLP/T-RFLP)
Comparative analysis of 16S rDNA has shown that highly conserved sequences
are interspersed with regions of variable sequences (Jayarao et al., 1992b). Analysis of
the variable regions can be used to determine the phylogenetic and evolutionary
relationships between bacteria. This technique can be used to determine strain specific
sequences between bacteria (Woese, 1987). Termination restriction fragment length
polymorphism analysis (T-RFLP) is used to analyse the variable sequence regions of
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