Denaturing Gradient Gel Electrophoresis Can Fail to Separate 16S rDNA Fragments with Multiple Base Differences

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments is commonly used to examine natural bacterial communities. However, recent studies have reported difficulty in separating different 16S rDNA sequences by DGGE. We utilized site-directed mutagenesis to create Escherichia coli 16S rRNA gene fragments differing by 1-4 base pairs, and examined the migration of these fragments in DGGE gels. DGGE could always separate sequences differing by a single base pair, but multiple sequence differences were not so easily resolved. Two sequences that differed by 2 base pairs showed identical migration in DGGE gels and could not be separated in a mixed sample. This limitation should be considered when using DGGE to examine natural bacterial communities.