M Beerens

DGGE FOR CARIES RISK ASSESSMENT IN ORTHODONTICS 3 45 of the reference species mixture ( R. dentacariosa, S. mutans, L. acidophilus, V. parvulla, A. nueslundii and L. plantarum ) was combined with 15 µl of loading buffer and loaded into wells flanking the plaque samples. Electrophoresis was performed at a constant voltage of 200 V at 60°C for approximately 4 h. The gels were stained with 4 µl of SYBR Gold (Invitrogen, Carlsbad, CA, USA) (11) in 200 ml TAE for 30 minutes with motion using a See-Saw Rocker (Bibby Scientific, Staffordshire, UK) at a speed (oscillator) of 5-10 Hz. After staining, the gels were placed on a UV transilluminator to visualise the band patterns per sample (Muyzer et al. , 1993). The visualised patterns were subsequently photographed with a digital camera (Canon Powershot G6, Tokyo, Japan). Plaque samples from 4 subjects were run in duplo on separate DGGE gels to test the repeatability of the method. DGGE band detection The DGGE gels were processed and analysed using GelCompar II software (version 6.5, Applied Maths, Sint-Martens-Latem, Belgium). Each gel contained 4 marker lanes to allow alignment of the band patterns. The same markers were used in all gels. A reference was defined from the markers on the first gel. All marker lanes on all gels were aligned using this reference to allow comparisons among gels. The banding patterns were normalised with respect to the marker lanes in all the gels. To determine optimum automated band detection settings, bands of the reference gel were first detected visually at three separate occasions two weeks apart. Then bands were searched by automated band detection using different combinations of settings for minimum profiling, grey zone, minimal area and shoulder sensitivity. The following settings were used: minimum profiling 0 to 6% absolute and 0 to 20% relative to the most intense band; grey zone settings 0 to 3; minimal area 0, 0.1 to 0.5 and 1 to 3; shoulder sensitivity 0 to 3. The optimum combination of settings for minimum profiling, both absolute and relative to the most intense band as well as visual detection of bands were then used on the full set of DGGE-gels. According to the manual of GelCompar II, minimum profiling relative to the most intense band is advised (“Gelcompar II manual version 6.5”, 2011).