Development of new procedures for the isolation of phytoplankton DNA from fixed samples

Abstract

Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples.The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results.

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

“…Other authors used a solution composed of 50 mM Tris, 20 mM EDTA, 0.5% Triton X100 and 0.2 mg ml -1 RNase (see, for instance, Bertozzini et al 2005) or employed the following buffer: 100 mM Tris, 50 mM EDTA, 100 mM NaCl (Wu et al 2000). …”

“…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).…”

Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

“…A high molecular variation has also been demonstrated for other nominal nanoflagellate taxa (e.g., for Spumella sp. [K. Pfandl et al, submitted for publication], Paraphysomonas vestita [2], Neobodo designis [33], and Rhynchomonas nasuta [22]). A conclusive judgment would require in-depth investigation of the respective morphospecies, which was, however, not in the scope of this study.…”

“…Most methods either require relatively large amounts of template DNA (i.e., cultured material, preserved tissues, or environmental DNA collected on filters or by centrifugation [18]) or amplification is limited to short fragments or both (2,4,6). It is therefore no coincidence that attempts to analyze the DNA sequence from preserved microplankton samples focused mainly on alveolate taxa, i.e., organisms presumably with a high copy number of the SSU rRNA gene (dinoflagellates [5,11,13,29]; ciliates [9]).…”

Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

“…This allows (close to real-time) prediction of the composition of the phytoplankton community before it becomes problematic (AlTebrineh et al, 2012;Anderson et al, 2012;Bertozzini et al, 2005). Some of the molecular methods include: fluorescent in situ hybridisation (FISH) (Touzet et al, 2010), fluorescent in situ hybridisation-flow cytometry (FISH-FC) (Eckford-Soper et al, 2013), enzyme-linked immunosorbent assay (ELISA), microarray for the detection of toxic algae (MIDTAL) (Medlin, 2013) and realtime qPCR (Penna and Galluzzi, 2013) The invention of PCR and qPCR technologies has vastly improved the analysis of nucleic acids from both quantitative and throughput perspectives.…”