Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences.
The phylogenetic affiliation of 91 operational taxonomic units, randomly sampled from three aquatic microcosm experiments, was investigated by two PCR based and one culture dependent method. The occurrence of multiple melting domains and poor coupling between Tm and DGGE retardation was demonstrated to cause poor resolution at the species level in PCR-DGGE analysis of microbial communities. We also showed that the problem of multiple melting domains was particularly prone for brackish water bacterioplankton in the Flavobacterium genus, providing characteristic band morphology for this genus. Banding patterns from DGGE analysis may therefore be misinterpreted in terms of the species richness in natural bacterial communities, when using commonly applied universal primers.
Kisand
Veljo
V
Umeå Marine Sciences Center, Umeå University, Norrbyn, SE-910 20, Hörnefors, Sweden.
Wikner
Johan
J
D001483:Base Sequence; D002999:Clone Cells; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006358:Hot Temperature; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D020560:Proteobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S