An improved method for DNA isolation from mucopolysaccharide-rich molluscan tissues

The use of molecular biology methods in studies of evolution and population genetics has increased dramatically during the last decade. Applications of DNA techniques in marine invertebrates in general and in molluscs in particular are affected by difficulties of DNA extraction of sufficient purity. This is due to large amount of polysaccharides in their tissues, which are usually isolated along with DNA. The polysaccharides can inhibit the activity of many molecular biology enzymes such as polymerases, ligases and restriction endonucleases1,2,3. Different methods have been proposed to overcome the problem: differential centrifugation4, high salt precipitation5, the use of benzyl chloride with repeated extraction6, Chelex resin7 or hexadecyltrimethylammonium bromide8, 9 (CTAB). The latter two methods are most common, but Chelex processed DNA is denatured and suitable only for the polymerase chain reaction (PCR)10. The substantial inconvenience of the CTAB approach is the necessity to keep the lysis buffer warm (at 60-65 ̊C) during the tissue grinding. It was reported that CTAB procedure sometimes results in partial DNA degradation5, 11. In this paper I propose an easy and inexpensive method of DNA extraction from fresh and preserved molluscan tissues that yields high-molecular-weight DNA suitable for virtually any kind of molecular biology studies. This method is an improved modification of the conventional DNA extraction protocol12. The procedure presented needs no special facilities and can be used in any laboratory. For this study I used molluscs from three major clades: 1) Polyplacophora (represented by Tonicella marmorea), 2) Gastropoda (Pulmonata—Cepaea sp., Prosobranchia—Margarites helicinus, Littorina saxatilis and L. littorea), 3) Bivalvia (Mytilus edulis). L. saxatilis, L. littorea and M. edulis were collected from the intertidal zone in the Kandalaksha Bay of the White Sea (August 1999), transported to the AlfredWegener-Institute (AWI) and kept alive in waterrecirculated aquaria (12°C, 25‰) before DNA extraction. T. marmorea and M. helicinus were obtained in the shallow waters at Spitzbergen coast by SCUBA diving (May 1999), tissues were frozen in liquid nitrogen and stored at -80°C until analysis. Cepaea sp. was collected in a park in the vicinity of the AWI (June 1999) and kept alive until DNA extraction. In T. marmorea and M. helicinus, the whole body was used for DNA extraction. In other molluscs, DNA was extracted either from foot muscles (L. saxatilis, L. littorea, and M. edulis) or separately from foot and hepatopancreas (Cepaea sp.). I consider the muscle tissue as the best source for DNA in molluscs. Digestive gland may contain parasites and hence the foreign DNA can contaminate a sample. 100–200 mg of muscle tissue was found to produce enough DNA for most applications. Before enzymatic treatment the tissue should be homogenized to ensure complete digestion. The usually recommended technique of grinding under liquid nitrogen often leads to DNA degradation13, pers. obs., and the use of a glass homogenizer is timeconsuming and not practical in studies which require processing of large amounts of animals (e.g. population studies). I routinely slice 50–70 mg of muscle on 2–3 mm thick pieces and squash them between two sheets of clean aluminum foil. The squashed tissue is added to a 2 ml plastic tube containing 1 ml of the lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, 1% sodium dodecyl sulphate (SDS), 0.2–0.4 mg/ml Proteinase K), briefly vortexed and incubated at 55oC until complete digestion. This usually takes 1–2 h depending on the size of tissue pieces. 100 l saturated KCl is added to the clear lysate and mixed well by repeated tube inverting (no vortex!). The solution is incubated on ice for 5 minutes. At this stage most of the polysaccharides and some proteins are precipitated along with the insoluble potassium dodecyl sulphate. Centrifuge the solution at maximal speed for 10–15 minutes, collect the supernatant in a clean tube and extract twice with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) mixture. Transfer the clear supernatant to another tube, add an equal volume of isopropanol, mix by inversion and incubate for 5–10 minutes at room temperature. Then centrifuge for 20 min at 15 000 g, discard the supernatant and wash the DNA pellet in 70% alcohol. Dry the pellet under vacuum and dissolve in 100 l of TE buffer (10 mM Tris-HCl, 1 mM EDTA). Add to the solution RNAse A (to the final concentration of 10 g/ml) and incubate at 37° for 30–60 min. The RESEARCH NOTES