Deionized formamide

The PCR products of the V3 region for the 16S rRNA genes were electrophoretically separated by DGGE using the Bio-Rad Dcode mutation detection system (Hercules, CA, USA) according to the manufacturer instructions. Next, 40 mL of each PCR product was loaded on 10% (w/v) polyacrylamide gels with a denaturing gradient from 30-60% and containing 12.6-25.2% (w/v) urea and 12-24% (v/v) deionized formamide (Sangon Biotech). Electrophoresis was performed in 1X TAE buffer for 10 min at a constant voltage 200 V at 60°C, and then at 85 V for 16 h. The gel was stained with 0.5 mL/mL ethidium bromide for 20 min, rinsed with deionized water for 20 min, and photographed. Characteristic bands were removed from the gel. The small gel pieces were rinsed with 100 mL 70% precooled ethanol 3-4 times, and then 50 mL double-distilled H 2 O was added to the gel pieces after they were air-dried and soaked for approximately 10 h at 4°C. PCR was conducted for the recovered DNA fragments under the same conditions as were used for the V3 region, and detected and purified by DGGE. Next, the recovered DNA fragments were amplified in the V3 region-specific primers F341 (5'-ACG GGG GGC CTA CGG GAG GCA G-3') and R518 (5'-ATT ACC GCG GCT GCT GG-3') without a GC-clamp. The PCR products were sequenced by Sangon Biotech.