Testing of primers for the study of cyanobacterial molecular diversity by DGGE.
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 3' or 5' 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.
Boutte
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C
Center for Protein Engineering, Institute of Chemistry B6, University of Liège, Belgium. c.boutte@student.ulg.ac.be
Grubisic
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S
Balthasart
P
P
Wilmotte
A
A
D000458:Cyanobacteria / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S