DGGE electrophoresis

DNA fragments with different sequences were separated in 8% polyacrylamide (acrylamide: bisacrylamide = 37.5:1; w/v) gels in 1X TAE buffer with 200 ng of each PCR product. A denaturing gradient of 40–60% was applied in the DGGE electrophoresis, formed with deionized formamide and urea. Gels were electrophorised in 1X TAE buffer at 60°C and 200 V for 3.5 h. Subsequently, the gels were washed with ultrapure water and stained with 5% GoldView™ dye for 30 min and photographed. DGGE graphs were digitized by Quantity One Analysis software (Gene Genius; Syngene, Frederick, MD, USA).