Algas

Plant Molecular Biology Reporter 18: 275–281, 2000 © 2000 International Society for Plant Molecular Biology. Printed in Canada.

Protocols

DNA Isolation Protocol for Red Seaweed (Rhodophyta)
RÉMI A. WATTIER*, PAULO A. PRODÖHL and CHRISTINE A. MAGGS
School of Biology and Biochemistry, The Queen’s University, Belfast, BT9 7BL, Northern Ireland, UK Abstract. We report a DNA isolation protocolfor red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 µg of high molecular weight DNA from 10 mg of driedmaterial, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species. Key words: DNA cloning, DNA isolation, genetic markers, polysaccharides (agarsand carrageenans), population genetics, red seaweed Red seaweed DNA isolation protocol Wattier et al.

Introduction Red seaweed is recognised as a species-rich phylum (> 2500 recognised species; Norton et al., 1996) with high commercial value as food and products such as hydrocolloids with over 2.5 billion $US annually (Radner, 1996). However, both the genome characterisation (Stoebe andKovallik, 1999) and intraspecific genetic biodiversity (Wattier et al., 1997; Engel et al., 1999) are still poorly known for many species. DNA isolation from red algae has proven very difficult (Sosa and Oliveira, 1992; Chesnick and Cattolico, 1993), mostly due to the co-isolation of hydrocolloids (agars and carrageenans). These compounds produce highly viscous solutions that may inhibit endonuclease andDNA polymerase activities (Jin et al., 1997). Polysaccharides can be efficiently removed using: i) CsCl gradient ultracentrifugation (Sosa and Oliveira, 1992; Chesnick and Cattolico, 1993), ii) agarose gel-electrophoresis purification (Saunders, 1993), or iii) hydroxyapatite column purification (Dutcher et al., 1990). While protocols with these steps provide high quality DNA, they are complex,time-consuming and require a large amount of
*

Author for correspondence. Present address: School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2DG, Wales, UK; e-mail: remi_wattier@hotmail.com; fax: (44) 01248 370731.

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material. None is compatible with the large number of samples associated with population-based studies. We report here atotal genomic DNA isolation protocol derived from a method originally developed for land plants (Dellaporta et al., 1983). Modifications were made to minimise polysaccharide co-isolation and to simplify the procedure for processing large numbers of samples. The protocol was applied to 12 red seaweed species and yielded DNA suitable for molecular cloning, endonuclease restriction and PCR. Material andMethods Plant material The 12 red seaweed species, the green alga, and the land plant species used to test the protocol are listed in Table 1. Seaweed samples were field collected from the northern coasts of Ireland in November 1998 and desiccated with silica gel (Sigma). Equipment

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1.5 mL microcentrifuge tubes Polypropylene pestles to fit 1.5 mL microcentrifugetubes Bottle with dispenser Water bath (37°C) with shaking High speed refrigerated microcentrifuge

Chemicals, solutions and buffers Stock extraction buffer: 100 mM Tris-HCl, 50 mM EDTA, 500 mM NaCl, pH 8.0. Autoclave and store at room temperature. SDS solution: 20% W/V in water. Store at room temperature. Proteinase K stock solution: 20 mg mL-1 in water. Store at -20°C. Ribonuclease A stock...