Homogeneous detection of cyanobacterial DNA via polymerase chain reaction (original) (raw)
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Journal of …, 1996
Oligonucleotide primers, specific for conserved regions of the genes encoding the β- and α-phycocyanin subunits of phycobilisomes (cpcB and cpcA) of cyanobacteria, were used to amplify a DNA fragment containing the intervening intergenic spacer region (cpcBA-IGS) of 19 strains of three morphospecies of cyanobacteria. Six Australian strains were identified as Anabaena circinalis Rabenhorst, six strains were identified as Microcystis aeruginosa Kützing, and seven strains were identified as Nodularia spumigena Mertens. Restriction enzyme digestion of the amplification products from the strains revealed restriction fragment length polymorphism (RFLP) within all three morphospecies. Strains corresponding to M. aeruginosa were highly polymorphic: 11 of the 14 restriction enzymes used displayed RFLPs. The A. circinalis and N. spumigena strains were less variable: three of 14 enzymes and seven of 14 enzymes, respectively, showed RFLPs. The presence of genetic variation between strains within these three divergent morphospecies, which span two orders of cyanobacteria (Chroococcales Wettstein and Nostocales (Borzi) Geitler), show that the cpcBA- IGS fragment has broad application as a molecular marker for intrageneric studies of cyanobacteria systematics and genetics.
An Improved Method for Marine Cyanobacterial DNA Isolation
World Journal of Microbiology & Biotechnology, 2005
The method of Bolch, Blackburn, Jones, Orr & Grewe [Phycologia, 36, 6 11, 1997] developed for isolation of DNA from freshwater cyanobacteria was suitably modified to yield a simple, efficient and reproducible protocol for the isolation of DNA from different morphological types of marine cyanobacteria. This method resulted in a high yield of quality DNA suitable for polymerase chain reaction (PCR) amplifications.
Nova Hedwigia, 2004
We examined 32 cyanobacterial strains, representative of 11 different genera, for the presence of the highly iterated genomic palindrome, called HIP1, using HIP1 extended PCR primers. Cryolysates or cell suspensions, and even single colonies, of Microcystis aeruginosa, strain PCC 7806, yielded PCR products equivalent to those obtained with purified DNA, as judged from the banding patterns obtained by gel electrophoresis. Cryolysates were used for amplification and genotyping of all other strains. Representatives of different genera differed extensively in HIP1-based migration patterns, and the amplification products of axenic cyanobacteria were comparable to those obtained with duplicate, non-axenic strains. In addition, HIP1 repeats were present in 11 strains of the species Microcystis aeruginosa and in isolates assignable to Planktothrix agardhii and P. rubescens. Based on available 16S rDNA sequence data and DNA/DNA hybridization results for members of these genera, the differences detected by HIP1-based profiling correlate with the interspecies distinctions between Planktothrix agardhii and P. rubescens, and reflect intraspecific diversity for members of Microcystis aeruginosa. Thus, we have confirmed the value of this method for rapid genotyping and verification of presumably identical strains from different culture collections.
Applied and Environmental Microbiology
For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other ...
Annals of RSCB NEW PRIMER COMBINATION FOR SEQUENCING THE CYANOBACTERIAL 16S rRNA GENE
In this study the possibility of sequencing the whole 16S rRNA gene with only three primers was analyzed. For this, we used genomic DNA from 11 cyanobacterial strains, 10 belonging to the genus Microcystis and one to the genus Nodularia. So far, the 16S rRNA sequence was obtained in our laboratory using 5 different primers or 6 when we wanted to sequence also the 16S rRNA – 23S rRNA Intergenic Transcribed Spacer (ITS). The fragments of interest were amplified by PCR, with several new primers combination. Not all the primers have specificity for cyanobacteria, but in order to avoid unspecific amplification, the DNA fragments were cloned in plasmidic vectors prior sequencing. The cloned amplicons were then bi-directionally sequenced, the obtained sequences were assembled and the entire 16S rRNA gene sequence was achieve. We amplified the 16S rRNA gene with 12 oligonucleotidic primers, in various combinations. Eventually, we succeeded to obtain the entire genic sequence with only three...
Molecular identification, typing and traceability of cyanobacteria from freshwater reservoirs
Microbiology-sgm, 2009
In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR-and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon-Wiener (J9) indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D50.83; J950.88) and Aphanizomenon flos-aquae the highest ones (D5J950.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.
Journal of microbiology (Seoul, Korea), 2006
Molecular characterization of ten marine cyanobacterial isolates belonging to the order Oscillatoriales was carried out using the phycocyanin locus (cpcBA-IGS) and the 16S-23S internally transcribed spacer region. DNA sequences from the phycocyanin operon discriminated ten genotypes, which corresponded to seven morphotypes identified by traditional microscopic analysis. The cpcB coding region revealed 17 % nucleotide variation, while cpcA exhibited 29 % variation across the studied species. Phylogenetic analyses support the conclusion that the Phormidium and Leptolyngbya genera are not monophyletic. The nucleotide variations were heterogeneously distributed with no or minimal informative nucleotides. Our results suggest that the discriminatory power of the phycocyanin region varies across the cyanobacterial species and strains. The DNA sequence analysis of the 16S-23S internally transcribed spacer region also supports the polyphyletic nature of the studied oscillatorian cyanobacteri...