( erase and primer pair 1055F ...)Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

erase and primer pair 1055F and 1406R (with GC-clamp), were resolved as discrete bands by DGGE. This primer set yielded two bands rather than one band for L. monocytogenes (Figure 1). One of the two bands migrated to the same location in the gel as the single band obtained from L. innocua and L. seeligeri. The second band produced from a 16S rDNA fragment from L. monocytogenes, which was not obtained from the other two species, migrated more slowly through the gel than the band common to all three species. The two bands suggest that L. monocytogenes has two 16S rRNA genes with slightly different sequences within the targeted region. This phenomenon is referred to as "sequence heterogeneity."⁹ It has long been known that bacteria have more than one gene coding for 16S rRNA to increase the rate of ribosome production in the cell. Normally the genes have the same sequence but for reasons not yet understood, some genes within the same organism have different sequences that, when transcribed, yield a ribosome with a slightly altered structure.

Alignment of the base sequences of the 16S rRNA molecules from the three Listeria spp. for the region corresponding to the gene fragment targeted by the primer pair 1055F and 1406R with GC-clamp reveals the 3 bases that differ in the fragment obtained from L. monocytogenes (Figure 2). This difference yields a DNA fragment with lower %G+C, melting at a lower denaturant concentration to produce a DGGE band that migrates a shorter distance through the gel than the bands produced from fragments from L. innocua and L. seeligeri. DGGE therefore appears to be quite sensitive to slight differences in base sequence.

Co-migration of gene fragme
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