M Beerens

44 CHAPTER 3 PCR (polymerase chain reaction) procedure The V2-V3 region of the 16S ribosomal DNA was amplified using the Muyzer primers F357 (5′- GC CGC CCG CCG CGC GGC GGG CGG CGG GGC GGG GGC ACG GGG CCT ACG GGA GGC AGC AG- 3′) and R518 (5′- ATT ACC GCG GCT GCT GG- 3′). Amplification reactions were performed in sterile 0.2 ml Eppendorf tubes using a PCR (polymerase chain reaction) thermocycler (Biometra, Göttingen, Germany). The reaction mixture, that was prepared in the PCR cabinet, consisted of PCR Buffer containing 1 μl of each primer, 1 μl dNTP’s, 1 μl BSA, 1.5 μl MgCl2, 2.5 μl 10× Taq Buffer, 0.5 μl HotStarTaqTM DNA polymerase (Qiagen, Hilden, Germany), 1 μl template DNA and sterile Milli-Q water to a final volume of 24 μl. The positive control contained S. mutans . The cycling parameters were: 35 cycles of 94 °C, 4 min (initial denaturation); 94 °C, 0.5 min (denaturation); 54 °C, 1 min (annealing) and 72 °C, 1 min (elongation); 1 cycle of 72 °C, 5 min (final elongation) and a holding temperature of 15 °C following the final cycle. PCR products (4.0 μl) were analysed by electrophoresis and then visualised on a 1.0% agarose gel stained with μl ethidium bromide. Bacterial DNA extracts from the reference species ( R. dentacariosa, S. mutans, L. acidophilus, V. parvulla, A. nueslundii and L. plantarum ) were separately PCR- amplified using the conditions described above. DGGE Denaturing Gradient Gel Electrophoresis DGGE was performed using the DCode universal mutation detection system (PowerPac basic BioRad TM , Hercules, CA, USA). The DGGE gels were prepared and run with 1X TAE buffer diluted from a 50X TAE buffer stock (2 mol/l Tris- base, 1 mol/l acetic acid and 50 mmol/l EDTA). The denaturing gradient was formed using two 8% acrylamide (ratio acrylamide: bis-acrylamide, 37.5:1) stock solutions containing low (30%) and high (70%) concentrations of urea and formamide, increasing in the direction of electrophoresis. A 0% denaturing solution contained only 40% bis-acrylamide. The gels were polymerised after adding 60 µl of a 10% ammonium persulphate (APS) solution and 12 µl of TEMED to the 30% and 70% denaturing solutions immediately prior to pouring the gradient gel. A total of 30 µl of APS and 6 µl of TEMED were added to the 0% solution and 60 µl of gel dye. The gel dye was used to enhance the visualisation of the wells during loading. The gels were polymerised for 2 h. The samples (20 µl) were mixed with 4 µl of loading buffer. On each gel, 10 µl