Dcode universal mutation detection system

DNA and RNA were extracted from the sourdough samples according to Doulgeraki et al. [27 (link)] in the first case and using the NucleoSpin^® RNAkit (Macherey-Nagel, Dueren, Germany) in the second. In the latter case, cDNA was synthesized using the PrimeScript^TMRT reagent kit (Takara, Kusatsu, Japan). As far as DNA and cDNA fragments are concerned, they were subjected to two PCR reactions. The approximately 250 nucleotides of the 5′ end of the 26S rRNA gene and the V6–V8 region of the 16S rRNA gene were amplified by PCR, in a final volume of 50 μL, using NL1 with a GC clamp and LS2 as primers in the first case and U968 with a GC clamp and L1401 in the latter one, in agreement with Paramithiotis et al. [30 (link),31 (link)]. PCR products were separated using the DCode Universal Mutation Detection System (Bio-Rad) with 8% (w/v) polyacrylamide gel containing urea-formamide (Applichem, Darmstadt, Germany) as denaturing agents in a concentration gradient from 20–60% in TAE buffer (40 mM Tris–acetate, 2 mM Na₂EDTA H₂O, pH 8.5). Electrophoresis took place at 50 V for 10 min and then 200 V for 4 h. Then, gels were visualized by ethidium bromide staining and photographed using a GelDoc system (Bio-Rad). Species identification was performed by co-migration with reference patterns.

For denaturing gradient gel electrophoresis genomic DNA extracted from landfill and marshland was amplified using primer 519FWD and 915GC which gave a product length of about 500 bp. DGGE was performed with a D-Code universal mutation detection system (Biorad, Hercules, CA, USA) using 16 cm by 16 cm and one mm gels. PCR products were loaded onto 7% (w/v) polyacrylamide gel. The polyacrylamide gels (Bis-Acrylamide, 37.5 : 1) were made with denaturing gradients ranging from 30 to 70%. 100% denaturant contained 7 M urea and 40% formamide. Electrophoresis was initially run at 200 V for 10 min at 60°C and afterwards for 15 h at 85 V. After electrophoresis, the gel was silver-stained and scanned under white light using Gel Doc (Biorad) (Figure 2). DGGE gel was further analyzed using Gel2K software (Svein Norland, Department of Biology, University of Bergen, Norway).

DGGE analysis was performed as described previously (McEwan et al. 2005 (link)). Briefly, analysis was performed as follows. Approximately 200 base pairs (bp) of the V3 region of the 16S rRNA gene were amplified using a forward primer: 5ʹ-TAC GGG AGG CAG CAG-3ʹ, and reverse primer: 5ʹ-ATT ACC GCG GCT GCT GG-3ʹ, with a GC-clamp (5ʹ-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC-3ʹ) at the 5′ terminus of the forward primer. PCR was performed using the following conditions: 5 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 60 °C, 1 min at 72 °C; 1 cycle of 1 min at 94 °C, 1 min at 60 °C, 10 min at 72 °C. Successful amplification was verified and the size of amplicons checked by agarose gel electrophoresis.
A DCode™ Universal Mutation Detection system (16 cm system, BioRad) was used to prepare and run the DGGE gels. DGGE parallel gradient gels ranged from 40 to 60% (8% acrylamide), and were run at 80 mV, 200 mA, for 16 h at 60 °C. DNA was visualised by staining with a DNA Silver Staining Kit (Amersham). In each case, all samples on a gel were from a single-organ source, with no comparisons made between gels.

The PCR extracts from bacterial isolates were sequenced by GATC-biotech company (Konstanz, Germany). Partial sequences were compared to those in the GenBank database using the BLAST program. Cheese PCR products were subjected to a TTGE analysis using a Dcode universal mutation detection system (Bio-Rad Laboratories, Hercules, CA, USA) following the method described previously [14 (link)].