Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs

Abstract

The ability of denaturing gradient gel electrophoresis (DGGE) technique to resolve 16S rDNA products generated from two different collections of bacteria using universal 16S primers was investigated. Alignments of 16S rDNA sequences of known species of rhizobia and methanotrophs were performed in order to determine the genetic variations within a 200 bp product obtained with PCR primers which amplify the 16S rRNA encoding genes from Eubacteria. Theoretical DNA melting curves were obtained with the Melt87 program and found to correlate with the ability to resolve fragments by DGGE. In the case of the rhizobia, the inability of DGGE analysis to resolve the PCR products from closely related species was in accordance with the low polymorphism observed amongst the sequences in the amplified area. In the case of the methanotrophs, the PCR products were surprisingly difficult to resolve given the high degree of sequence polymorphism of the amplified area in some distantly related species. The difference in sequence divergence within the two groups members allowed therefore to scale the resolution ability of the DGGE technique.

Bacterial diversity from the source to the tap: a comparative study based on 16S rRNA gene-DGGE and culture-dependent methods

“…DGGE, in spite of its bias and all its limitations, including low coverage (Muyzer et al, 1993;Van Wintzingerode , 1997;Muyzer & Smalla, 1998;Farnleitner et al, 2004), is an adequate method when the aim is to compare patterns along a process or over different sites (Gilbert et al, 2012;Ling et al, 2012;Yang et al, 2012). Although DGGE may also be used to assess phylogenetic diversity, a major drawback is the poor resolution, when different nucleotide sequences co-migrate in a single band, violating the rule one-band-one-organism assumed in this technique (Vallaeys et al, 1997). Nevertheless, it is possible through band cloning to overcome such a difficulty.…”

Decreased colonization of fecal Clostridium coccoides/Eubacterium rectale species from ulcerative colitis patients in an in vitro dynamic gut model with mucin environment

“…However, the number of bands in a DGGE does not necessarily reflect the actual diversity of the microbial community. One band may represent several species with identical partial 16S rRNA gene sequences (Vallaeys et al, 1997), but on the other hand, one species may also produce more than one band because of multiple, heterogeneous rRNA operons (Dahllof et al, 2000). The richness analysis must, therefore, be interpreted as an indication rather than an absolute measure of the microbial diversity.…”

Spatial comparison of total vs. active bacterial populations by coupling genetic fingerprinting and clone library analyses in the NW Mediterranean Sea

“…Comigration of PCR products from different species within the same peak is a common limitation of fingerprinting methods (Kowalchuk et al , 1997; Vallaeys et al , 1997; Piceno et al , 1999; Casamayor et al , 2000). In our study, we checked this constraint in detail by running the CE‐SSCP signature of 80 individual clones in parallel to the natural complex assemblage.…”