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gene mixtures and hence can be used to assess diversity within a simple bacterial
community (Dunbar et al., 2000).
1.6.3.5 -
Amplified fragment length polymorphism (AFLP)
AFLP involves the digestion of total purified genomic DNA using a restriction
endonucleases (Vos et al., 1995; Janssen et al., 1996; McLauchlin et al., 2000:
Rademaker et al., 2000). The digest products are then ligated to a double stranded
oligonucleotide adapter which is complementary to the base sequence of the restriction
site (Zabeau & Vos, 1993). Selective PCR of the fragments is achieved using primers
which correspond to the adapter/restriction site sequence. These sequences are then
analysed by gel electrophoresis (McLaughlin et al., 2000; Zhao et al., 2000). This allows
characterisation of the bacterial isolates under investigation to the species, sub-species
and strain level (Rademaker & de Bruijn, 1997).
1.6.3.6 -
AP-PCR and RAPD
Arbitrarily primed PCR (AP-PCR) (Welsh & McClelland, 1990) and random
primed amplified polymorphic DNA (RAPD) (Williams et al., 1990) are based on the
amplification (using the polymerase chain reaction) of multiple DNA fragments from
genomic DNA with random or arbitrary primers. This results in a characteristic pattern of
amplicons for each strain of a given species. Thus these techniques permit the
differentiation of isolates to the species, subspecies and strain level.
1.6.3.7 - rep-PCR, ERIC-PCR and BOX-PCR
These techniques rely on the amplification of conserved repetitive sequences
found within the intergenic regions at sites dispersed throughout the genome of bacteria
(Versalovic et al., 1994). Such regions of noncoding, repetitive sequences can be used for
multiple genetic targets for oligonucleotide primers, enabling the generation of unique
DNA profiles or fingerprints for individual bacterial strains (Versalovic et al., 1994;
Rademaker & de Bruijn, 1997; Louws et al., 1998; Rademaker et al., 1998; Rademaker et
al., 2000). ERIC-PCR utilises amplification of the enterobacterial repetitive intergenic
consensus, rep-PCR utilises the repetitive extragenic palindromes and BOX-PCR
amplifies a BOX element found mainly in Gram positive bacteria (Tas & Lindström,
2000). The genomic fingerprints produced using these techniques allow the determination
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