Testing of primers for the study of cyanobacterial molecular diversity by DGGE

Abstract

Denaturing Gradient Gel electrophoresis (DGGE) is a PCR-based technique which is widely used in the study of microbial communities. Here, the use of the three specific 16S rRNA cyanobacterial specific primers CYA359F, CYA781R(a) and CYA781R(b) on the assessment of the molecular diversity of cyanobacterial communities is examined. Assignments of the reverse primers CYA781R(a) and CYA781R(b) with cyanobacterial strain sequences showed that the former preferentially targets filamentous cyanobacteria whereas the latter targets unicellular cyanobacteria. The influence of the GC clamp position on the forward or on reverse primer and the use of the two reverse primers separately or in equimolar mixture were investigated. Three environmental samples were subjected to amplification with 6 combinations of primers. The 6 banding patterns as well as the sequences of the bands extracted were analysed and compared. In addition, to assess the effect of the position of the GC clamp, the melting profiles of the sequences of Aphanizomenon flos-aquae PMC9707 and Synechococcus sp. MH305 were determined, with the GC clamp in the 3V or 5V position. Results showed that the use of two separate amplifications allowed a more complete study of the molecular diversity of the cyanobacterial community investigated. Furthermore, similar richness and identical phylogenetic assignments of extracted bands were obtained irrespective of the positioning of the GC clamp. D

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

“…Approximately 450 bp of the 16S rRNA gene fragments (corresponding to Escherichia coli positions 358 to 805) were amplified using two sets of cyanobacterium-specific primers (20): primer pair CYA359F (with a 20-nucleotide GC clamp at the 5= end) and CYA781R(a) and primer pair CYA359F (with a GC clamp) and CYA781R(b), as described by Boutte et al (21). Thus, two PCR products were amplified for each sample and run on separate lanes in TGGE.…”

Diversity of planktonic cyanobacteria and microcystin occurrence in Polish water bodies investigated using a polyphasic approach

“…DNA extraction and 16S rRNA PCR-DGGE: DGGE analyses targeting 480 pb of the V3 and V4 regions of 16S rRNA were performed as described by Boutte et al (2006) DGGE dendrogram: Two DGGE fingerprints (a) and (b) were obtained from each sample and analysed using GelCompar II Software 2.5 (Applied Maths). The bands were localised on the basis of densitometric curves.…”

Heavy Metals Pollution Influence the Community Structure of Cyanobacteria in Nutrient Rich Tropical Estuary

“…The morphological methods mainly depend on the size and pigmentation of cyanobacteria, which may vary with the nutrient compositions while culture dependent method is limited by the lack of a universal media which can accommodate all cyanobacteria. In PCR-DGGE, the PCR product of each species from the sample is segregated into distinct bands in a gradient gel [49]. Hence each band in a gradient gel can be considered as an operational taxonomic unit (OTU).…”

“…Here we used three 16S rRNA gene primers specific for cyanobacteria Cya359F, Cya781R (a) and Cya781R (b), for the assessment of community structure of cyanobacteria in CE. The reverse primer Cya781R(a) preferentially targets filamentous cyanobacteria whereas Cya781R(b) targets unicellular cyanobacteria [49]. Interestingly, the 16S rRNA gene sequence derived from majority of bands (13 number) were matched with previously uncultivated cyanobacteria sequence data available in NCBI.…”