An efficient DNA isolation protocol for filamentous cyanobacteria of the genus Arthrospira

Abstract

Thanks to their photosynthetic and nutritive properties, cyanobacteria of the Arthrospira genus are of interest as food supplements, as efficient oxygen producing life support system organisms for manned space flight, and for the production of biofuels. Despite these potential valuable applications, full genome sequences and genetic information in general on Arthrospira remain scarce. This is mainly due to the difficulty to extract sufficient high molecular weight nucleic acids from these filamentous cyanobacteria. In this article, an efficient and reproducible DNA extraction procedure for cyanobacteria of the genus Arthrospira was developed. The method is based on the combination of a soft mechanical lysis with enzymatic disruption of the cell wall. The comparison with other extraction protocols clearly indicates that this optimised method allows the recovery of a larger amount of DNA. Furthermore, the extracted DNA presents a high molecular weight, a reduced degradation and an excellent overall quality. It can be directly used for molecular biology purposes such as PCR, and clone library construction.

An improved method for efficient isolation and purification of genomic DNA from filamentous cyanobacteria belonging to genera Anabaena, Nodularia and Nostoc

“…This amount (see Table 1) is similar to (for Nostoc) or higher than (for Anabaena and Nodularia) values reported by other authors who used different, phenol-based methods (Fiore et al 2000, Wu et al 2000. Moreover, these results confirm previous observations (Fiore et al 2000, Morin et al 2010) that any method devoted to cyanobacterial DNA isolation and purification, reported to date, varies considerably in its efficiency from one genus (or even strain) to another.…”

“…To assess the purity of DNA samples, we have determined A260/A280 and A260/A230 ratios. It is generally accepted that a DNA sample of high purity should have the A260/A280 ratio of about 1.8 and the A260/A230 ratio between 2.0 and 2.2 (Manchester 1995, Sambrook and Russell 2001, Morin et al 2010. The A260/A280 value below 1.8 indicates a possible protein contamination, while the A260/A230 value below 2.0 or above 2.2 indicates the presence of carbohydrates, phenols or other aromatic contaminants.…”

“…All these compounds interfere with commonly used procedures of DNA isolation and purification, which results in obtaining samples containing small amounts of highly contaminated DNA, usually useless in molecular cloning procedures (Wu et al 2000). Commercially available DNA purification kits, based on binding of nucleic acids to silicon membranes at high concentrations of chaotropic salts, are usually useless if one needs to isolate genomic DNA from filamentous cyanobacteria (Bertozzini et al 2005, Morin et al 2010. Although some procedures of DNA isolation and purification from these organisms were reported, the serious problem is that usually their efficiencies vary considerably from one species to another (Fiore et al 2000).…”

“…Although some procedures of DNA isolation and purification from these organisms were reported, the serious problem is that usually their efficiencies vary considerably from one species to another (Fiore et al 2000). Therefore, it appears that optimization of specific methods separately for particular genus or a group of a few genera is necessary (Morin et al 2010). Most procedures ensuring elimination of contaminating compounds present in cyanobacterial envelopes cause DNA lesions, therefore, a genetic material thus isolated may be inappropriate for construction of genomic libraries.…”

“…In the light of these problems, we aimed to develop an improved method for efficient isolation and purification of genomic DNA from filamentous cyanobacteria. Since specificity of particular methods to certain genera of filamentous cyanobacteria has been demonstrated previously (Fiore et al 2000, Morin et al 2010, in this study we have focused on the genera Anabaena, Nodularia and Nostoc. This choice was based on the fact that these cyanobacteria appear to be a potential source of bioactive compounds, while reports on effective DNA isolation from them are scarce.…”