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Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

how the DCode system was used to detect a unique base pair sequence in a fragment of the16S rDNA of L. monocytogenes amplified by PCR using a general primer set and the Gene CyclerTM. The approach permitted detection of L. monocytogenes in the presence of two closely-related species, L. innocua and L. seeligeri.


Methods and Materials
THEORY OF DGGE
DGGE is a technique that separates DNA fragments of the same length but with different guanine + cytosine (G+C) base content. Separation is based on the concentration of a denaturant such as urea or formamide needed to effect separation (melting) of double-stranded helical fragments into partially-separated, single-stranded fragments. The G+C content of the fragment determines the denaturant concentration at which domains of the fragment will melt. A partially-melted fragment migrates more slowly through a polyacrylamide gel than does an undegraded, helical, doublestranded DNA molecule. The denaturation reaction can be controlled by establishing a gradient of DNA denaturants such as urea and formamide within the gel. During electrophoresis, the migrating double-stranded fragment begins to melt upon exposure of various domains of the fragment to an increasing denaturant concentration. Domains with a low %G+C will melt at a low denaturant concentration, while domains with high %G+C melt at higher denaturant concentrations further down the gel.

A 40 base pair (bp), GC-rich oligonucleotide referred to as a GC-clamp can be attached to the fragments using cloning techniques to retard migration of melted fragments through the gel to achieve better resolution of fragments with small differences in sequence variati
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