The PCR products with a GC-clamp (20e25 ml) were separated using DGGE with modifications in the denaturant gradient and acrylamide-bisacrylamide concentration (9%). The gels were run for 17.5 h at 80 V. After electrophoresis, the gels were stained with SYBR Gold nucleic acid gel stain and photographed. The PCR fragments without a GCclamp were cloned into commercial plasmid vectors