DNA extraction from sea anemone (Cnidaria: Actiniaria) tissues for molecular analyses

A specific DNA extraction method for sea anemones is described in which extraction of total DNA from eight species of sea anemones and one species of corallimorpharian was achieved by changing the standard extraction protocols. DNA extraction from sea anemone tissue is made more difficult both by the tissue consistency and the presence of symbiotic zooxanthellae. The technique described here is an efficient way to avoid problems of DNA contamination and obtain large amounts of purified and integral DNA which can be used in different kinds of molecular analyses.


INTRODUCTION
Sea anemones (Anthozoa: Actiniaria) are simple askeletal polyp animals, and a very diverse, ecologically important group of organisms (Schick, 1991). In spite of the many techniques that have been developed, the isolation of template DNA for PCR (Saiki et al., 1988) amplified from individual, small organisms can be difficult. Recently, DNA-based techniques have been used to determine the relationship of the Cnidaria to other metazoa and to examine relationships within the Cnidaria (e.g., Hori and Satow, 1991;Christen et al., 1991;Bridge et al., 1992;1995;Chen, et al., 1995;Beagley et al., 1995;Veron et al., 1996;France et al., 1996;Odorico and Miller, 1997;Romano and Palumbi, 1997). Such approaches are elucidating long-standing controversies about relationships within the Cnidaria.
This article reviews the published literature and suggests a feasible DNA extraction method to obtain large amounts of pure and integral DNA for molecular analyses.

DNA extraction
We unsuccessfully attempted to extract DNA from sea anemones using previously published protocols. We then optimized the protocols by including some important additional steps involving careful adjustments in tissue maceration, followed by optimization of the concentrations of proteinase K and by modification of the standard phenol:chloroform extraction techniques.
Total genomic DNA was extracted from pieces of 100% ethanol-preserved sea anemones. For most specimens small pieces (approximately 0.5 to 1.0 cm) of tissue from the pedal disc were used, which avoided possible amplification of the zooxanthellae DNA (Fautin and Smith, 1997). In all steps, shaking was for 15 min and centrifugation for 4 min at 6500 rpm, except in the third step that centrifugation was at 13.000 rpm. Prior to extraction the tissue fragments were placed in an Eppendorf tube and incubated at 35ºC for 2 h in order to withdraw excess etha-METHODOLOGY DNA extraction from sea anemone (Cnidaria: Actiniaria) tissues for molecular analyses S.M. Pinto 1 , F.M.C. Fernandes-Matioli 2 and E. Schlenz 1 Abstract A specific DNA extraction method for sea anemones is described in which extraction of total DNA from eight species of sea anemones and one species of corallimorpharian was achieved by changing the standard extraction protocols. DNA extraction from sea anemone tissue is made more difficult both by the tissue consistency and the presence of symbiotic zooxanthellae. The technique described here is an efficient way to avoid problems of DNA contamination and obtain large amounts of purified and integral DNA which can be used in different kinds of molecular analyses. nol. In step one, small pieces of tissue were ground into powder under liquid nitrogen and then placed in a final volume of 400 µl of lysis buffer (10 mM Tris-HCL, pH 8.0, containing 0.25 M EDTA, pH 8.0, 2% (w/v) sodium dodecyl sulfate, SDS). In step two, RNAse was added to a final concentration of 0.6 mg/ml and the sample was incubated for 30 min at 37ºC in order to eliminate any RNA molecules. After that, proteinase K was added to a final concentration of 1 mg/ml and the mixture incubated for 72 h at 37ºC. After incubation an equal volume of phenol was added to the sample, which was then shaken and centrifuged. Approximately 360 µl of the supernatant solution was then transferred to a clean tube and an equal volume of phenol added. Shaking and centrifugation were performed using the same conditions as above and 340 µl of the supernatant was transferred to a new tube with an equal volume of chloroform:isoamylalcohol (24:1) and gently shaken before centrifugation. In step three, 300 µl of the supernatant was transferred to a new tube containing 30 µl of 6 M NaCl and gently stirred. The DNA was precipitated with 2.5 volumes of very cold absolute ethanol and the samples centrifuged for 4 min at 13.000 rpm. The supernatant was drained off and the pellet was washed with 70% ethanol and air-dried for 24h with the tube inverted. The samples were resuspended in an appropriate volume of TE buffer (10 mM Tris-HCL, pH 8.0, and 1 mM EDTA) and incubated at 37ºC for at least 48 h, until completely soluble.
DNA was quantified in a spectrophotometer (Itachi model U-2000), the mean purity value was 1.8 ± 0.1 and extracted DNA concentration was 300-1.400 ng/µl. The integrity of the extracted DNA was checked using 2 µl of each sample on a 1.0% agarose gel stained with ethidium bromide. Figure 1 shows that the samples presented no signs of degradation. Figure 1 (upper half, lanes 6-9) also shows four unsuccessful DNA extractions, where samples were incubated (24 h at 37ºC) in the presence of proteinase K.
DNA extracted from sea anemone samples was used as a template in PCR amplifications with the SPAR technique (Gupta et al., 1994). The tetranucleotide repeat primers used were 16 bases long. A variety of primers were assayed with the most informative outcomes from (GACA) 4 .
In a study of the population structure of Actiniaria species being undertaken by S.M. Pinto and her colleagues, (GACA) 4 is being used as a molecular marker under the name Micro 5. cler for 35 cycles. The first 5 cycles consisted of 45 s at 94ºC, 60 s at 51ºC, and 60 s at 72ºC. The final 30 cycles consisted of 45 s at 92ºC, 60 s at 48ºC, and 60 s at 72ºC. All products were visualized on a 1.4% agarose gel stained with ethidium bromide (Figure 2). Negative controls lacking sea anemone DNA were included in PCR assays to monitor any possible contamination (Palumbi, 1996).

DISCUSSION
The systematics of sea anemones is currently based on only a few morphological characters (see McCommas, 1991;Fautin and Smith, 1997), and may potentially improve the use of molecular characteristics for clarifying phylogenetic relationships among genera and species. The utility of DNA molecular markers for addressing evolutionary questions in sea anemones has already been demonstrated by Pont-Kingdon et al. (1994), Beagley et al. (1996), Finnerty and Martindale (1997), and Fautin and Smith (1997.
The results of our study show the striking effects of some details on the success of the DNA extraction protocol. The tissue type, maceration and digestion of tissue and amount of time allowed for proteinase K incubation were all essential for obtaining high-quality DNA.
Tissue type has been found to affect the success of DNA analyses of samples from plants (Chase and Hillis, 1991;Rogstad, 1992), birds (Seutin et al., 1991) and insects (Altschmied et al., 1997). We concluded that the dehydration of tissue until there is no trace of ethanol was the best solution to avoid interference in the reactions occurring during the extraction. Given the hard consistency of the tissue the use of liquid nitrogen to homogenize the samples was important to the success of DNA extraction. Chen et al. (1995) and Strassmann et al. (1996) state that grinding in liquid nitrogen is a simple and efficient means of isolating significant amounts of high-quality genomic DNA from tissues.
Duration and temperature of incubation with proteinase K also influenced the success of this method, and high incubation temperatures were inefficient in our study, although the temperature employed in many studies have ranged from 50º to 65ºC (McMillan et al., 1988;ten Lohuis et al., 1990;McMillan et al., 1991;Coffroth et al., 1992;Chen et al., 1995;France et al., 1996;Romano and Palumbi, 1997;Lopez and Knowlton, 1997;Lopez et al., 1999). Pilot experiments using a final concentration of 1 ng/ml of proteinase K at 37ºC for 24-48-72 h revealed that the tissues were totally digested after 72 h. Low concentrations of proteinase K failed to digest tissue at any temperature and incubation time. We, therefore, opted for a slow and gradual digestion method because of the hard consistency of the tissue.
To avoid any protein residues, we used double washes of phenol followed by repeated extractions with chloroform:isoamylalcohol (24:1). Since the intermediate phenol/chlorofom/isoamylalcohol steps described by Chen et al. (1995) did not affect the final quality of the samples these were omitted from our protocol.
The gradual decrease of the volume of recovered supernatant (400/360/340/300 µl) guaranteed the high quality of the extracted product. After washing in 70% ethanol, DNA pellets are best dried by air for at least 24 h or dried in a vacuum centrifuge for 2-5 min. This step is particularly important for the success of PCR and sequencing reactions and prevents DNA degradation.
Samples of pure DNA are invaluable in studies of genetic evolution and systematic phylogenetic approaches using molecular data. The integrity of DNA is especially important for the amplification of large fragments. Figure 1 shows no signs of DNA degradation in the samples so that they could be used to amplify large fragments. High concentrations of extracted DNA (300-1400 ng/µl) have allowed us to begin a genetic stock of several species of sea anemone. The method described in this paper is a feasible method which allows the isolation of good DNA from tissues of sea anemones, and the protocol may also be applicable to other fleshy marine cnidarians.

ACKNOWLEDGMENTS
We are most grateful to Dr. L.F. de Almeida-Toledo (Universidade de São Paulo (USP), São Paulo), who has facilitated our study in the Laboratório de Ictiogenética no Instituto de Biociências da USP. We are also grateful to Dr. S.L. Romano (University of Guam, EUA) and Dr. S.R. Matioli (Universidade de São Paulo (USP), São Paulo) for critically reviewing the manuscript. We are indebted to Dr. A. Excoffon and P. Braga (Ciencias Marinas, Facultad de Ciencias Exactas y Naturales. UNMdP, Mar del Plata, Argentina) for collecting of specimens of A. chilensis and T. errans. Thanks are also due to M. Rivadeneira (Universidad de Chile) for the donation of specimens of A. chilensis. Financial support was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil, by a grant to the first author (No. 97/12232-9). Publication supported by FAPESP.