GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION¹

Abstract

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μg genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A260/A280 ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum. The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.

Dinucleotide microsatellite markers in the genus Caulerpa

“…Specific primers were designed for 18 sequences containing dinucleotide repeats from the libraries previously developed by Varela-Álvarez et al (2006). Ten loci were found to be polymorphic in at least one species.…”

“…DNA was purified by isolation of nuclei. Construction and screening of the genomic libraries are described in Varela-Álvarez et al (2006), based on small modifications of protocols from Billote et al (1999) and Kijas et al (1994). A total of 18 sequences obtained from these libraries containing microsatellite repeats were used to design primers using PRIMER 3 (Rozen and Skaletsky 2000).…”

A novel anticoagulant peptide from the Nori hydrolysate

“…The genomic DNA was isolated by CTAB method (Varela-Alvarez & Andreakis, 2006). To design primers, sequences for the following species were retrieved from GenBank, viz.…”

A comprehensive cDNA library of light- and temperature-stressedSaccharina latissima(Phaeophyceae)

“…A novel protocol had to be established for RNA extraction because standard procedures proved inapplicable to brown alga, due to the high amounts of polysaccharides (Wang et al, 2005;Varela-Alvarez et al, 2006) and phenolic compounds (Lane et al, 2006;Pearson et al, 2006). The new method (see Materials and methods) yielded 16.53 AE 4.99 mg RNA per 250 mg of frozen tissue and the extracted RNA was of high purity, showing A 260/280 ratios of 2.17 AE 0.04 and A 260/230 ratios of 2.23 AE 0.22 (values are means AE SD, n ¼ 240).…”