OPTIMIZATION OF DNA EXTRACTION FROM BROWN ALGAE (PHAEOPHYCEAE) BASED ON A COMMERCIAL KIT¹

Abstract

Large-scale DNA molecular studies require reliable and efficient tools for DNA extractions. However, for some plant species and brown algae, isolation of high-quality DNA is difficult. We developed a novel method for isolating high-quality DNA from the polysaccharide-rich and polyphenol-rich brown algae based on a commercial kit and protocol (Qiagen) by optimizing the lysis step and including a chloroform ⁄ isoamyl alcohol supplementary purification step. DNAs from 24 brown algal species extracted using the original and the modified Qiagen protocol were compared for yield, quality, and effectiveness in PCR amplification. There was no significant difference in the yields between protocols. However, a statistically significant increase in DNA purity was obtained with the modified protocol, for which the A 260 ⁄ A 280 and A 260 ⁄ A 230 absorbance ratios averaged 1.66 ± 0.05 and 1.31 ± 0.01, respectively, compared to 1.37 ± 0.04 and 0.52 ± 0.04 with the original protocol. DNAs extracted by the modified procedure were more successfully amplified by PCR (nuclear, mitochondrial, and chloroplastic regions) than DNAs extracted using the original commercial kit and protocol. Importantly, the modified protocol can be applied in a high-throughput (e.g., 96-well plate) format, allowing a higher efficiency for downstream molecular analysis. In addition, improved DNA quality could increase its stability for long-term storage.

Species Diversity, Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae, Dictyotales)

“…DNA extraction, amplification and sequencing. Total genomic DNA was extracted from tissue samples dried in silica gel using an extraction protocol detailed by Snirc et al (2010). Three organellar protein-coding regions were PCR-amplified.…”

A multi-locus time-calibrated phylogeny of the brown algae (Heterokonta, Ochrophyta, Phaeophyceae): Investigating the evolutionary nature of the “brown algal crown radiation”

“…Total genomic DNA was extracted with a modified DNeasy Plant MiniKit protocol (Qiagen, Hilden, Germany), according to the brown algae specific protocol developed in Snirc et al (2010). Sequences of the five mitochondrial genes cox1, cox3, nad1, nad4 and atp9, and of the four plastid genes rbcL, psaA, psbA and atpB, were amplified and sequenced.…”

Phylogenetic Analysis of Phyllospadix iwatensis Based on Nucleotide Sequences Encoding 18S rRNA and ITS-1

“…Methods have been described for the successful isolation of genomic DNA from various macroalgal species (Nakajima et al, 2000;Snirc et al, 2010;Hoarau et al, 2007). In this study, genomic DNA was isolated from ground Phyllospadix using the STE and CTAB buffer method (Valera-Alvarez, 2006).…”