Isolation of DNA from tannin-containing plants

Plant Science Letters, 11 (1978) 323--328 © Elsevier/North-Hollano Scientific Publishers Ltd.

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ISOLATION OF DNA FROM TANNIN-CONTAINING PLANTS* DIANA B. STEIN** and WILLIAM F. THOMPSON

Department of Botany, University of Massachusetts, Amherst, Mass. 01003 (U.S.A.) (Received August 5th, 1977) (Revision received and accepted November 3rd, 1977)

SUMMARY

A combination of techniques has been developed which permits purification of large amounts of DNA from plants containing high levels of tannins and brown pigments. The method uses lyophilized plant tissue, permitting storage Of field collections and eliminating the need for fresh material. The tissue is disrupted by blending at 60 ° C. The DNA is separated by gel fractionation followed by hydroxyapatite (HAP) chromatography and the final product is concentrated by lyophilization. In this procedure all enzyme treatments and precipitation steps may be avoided.

INTRODUCTION

Extraction of large amounts of highly purified DNA from many higher plants may be complicated b y the presence of tannins and brown pigments. Such compounds are often produced during tissue disruption when endogenous phenolics become oxidized. Anderson [1] has reviewed many methods for inhibiting such polyphenol oxidase activity during the isolation of enzymes and/or organelles. Some of these methods as well as other approaches have been adapted to separate nucleic acids from pigments. For example, Stern [2] noted that after traditional DNA isolation procedures were applied to plants, colored contaminants might still remain and could be removed by Sephadex G-50 chromatography. Gusta and Runeckles[3] suggested more drastic procedures involving the use of dimethyl sulfoxide at temperatures of * C.I.W.--D.P.B. Publication No. 617. ** Present Addre~es: (D.B.S.) Department of Zoology, University of Massachusetts, Amherst, Mass. 01008; (W.F.T.) Department of Plant Biology, Carnegie Institution of Washington, 290 Pa~imn Street, Stanford, Calif. 94395 (U.S.A.) Abbreviations: DIECA, diethyl dithiocarbamate; HAP, hydroxyapatite; PVP, polyvinylpyrrolidone; SDS, sodium dodecyl sulfate.

324 --40 ° C to remove nucleases and pigments prior to nucleic acid extraction from apple bark. Ferns and fern allies have proven to be especially difficult material for nucleic acid isolation since crude extracts of these species often contain unusually high levels of tannins, and only very low yields of DNA have been reported. Green [4] used DIECA to inhibit polyphenol oxidase activity and to prevent binding of polymerized phenolics to the DNA, and then applied standard enzyme treatments and alcohol precipitations. However, even though the DNA could be fully purified, the yields were generally low. A. Bendich (personal communication) has isolated larger amounts of DNA from ferns and horsetails by using PVP to bind phenolics and DIECA to inhibit polyphenol oxidase, but these methods were insufficient when applied to the Osmundaceous ferns. The crude extracts always contained large amoun.ts of brown pigments, and alcohol precipitates from such preparations were highly resistant to further purification. We describe here a method of DNA isolation developed for these ferns which we believe will be of general utility. The technique uses lyophilized plant material, thus permitting convenient tissue storage, and allows recovery of large amounts of purified DNA from crude extracts containing high levels of tannins and brown pigments. MATERIALS AND METHODS Three native species of Osmunda were used in this study: Osmunda regalis, O. cinnamomea, and O. claytoniana. Only fully expanded leaves were used since preliminary work with equal weights of "fiddlehead" and mature leaf tissue gave lower yields of DNA from the former. B.R. Green (personal communication) observed a similar reduction in DNA yield from other fern croziers and suggested that this tissue may contain an active DNAase. Mature fronds were collected from field populations in western Massachusetts. Nonfertile pinnae were removed from the rachis, cut into 2--3-cm pieces, wrapped in cheesecloth, and frozen in liquid nitrogen. The tissue was lyophilized and subsequently stored at room temperature in tightly sealed jars containing Drierite (anhydrous CaSO4). Extraction media were freshly prepared on the day of use since DIECA decomposes rapidly. The medium contained 10 mM Na2 EDTA, 300 mM NaC1, 100 mM Tris, pH 8.4 (at 25 ° C), 2.5% (w/v) DIECA, 2.5% (w/v) Polyclar AT (PVP, GAF Co.) and 1% (w/v) SDS. Lyophilized tissue was homogenized in a Waring Blendor with extraction buffer (100 ml per 4 g dry wt) which had been preheated to 60 ° C. After an additional 30 rain at 60 ° C, the homogenate was centrifuged at 20 000 × g for 20 rain at 0--4 ° C. The supernatant fractions were adjusted to 1 M with respect to NaCIO4, shaken with an equal volume of chloroform/octanol (24 : 1) and the aqueous phase recovered after centrifugation at 20 000 X g for 20 min. Chloroform/octanol extractions were repeated until all chlorophyll was removed and only minor amounts of denatured protein appeared at the interface.

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Fig. 1. Elution profile of a concentrated crude extract of O. regalis on a Bio-Gel A-50m column (4 x 50 cm) as determined by diphenylamine assay. The volumes for the first 26 fractions were 9.3 ml; the remainder were 12.0 ml. Colors o f the fractions are indicated to illustrate retardation of the tannins. An aliquot o f each fraction was tested with the diphenylamine reagent and the Asps--A6s o was measured to locate the DNA.

The crude extract, yellow to brown in color (depending on the species) was concentrated by dialysisagainst Aquacide I(polyethylene glycol, tool. wt. ca. 70 000, Calbiochem) at 4 ° C until the volume was reduced to 25--40 ml. The extract was then dialyzed extensively against 100 m M NaCl, 10 m M Tris, p H 7.5 at 25 ° C and clarifiedby centrifugation at 20 000 X g. The clarified sample was applied to a 4 × 50 c m column (bed volume of 625 ml) of Bio-Gel P-200 or Bio-Gel A - 5 0 m (Bio-Rad) equilibrated with Tris--NaC! buffer at 4 ° C. The fractions containing D N A (void volume) were located by using Burton's [5] diphenylamine assay (Fig. I). A trace of brown color sometimes eluted with the exclusion peak, but the majority of the pigments were greatly retarded. The nucleic acid-containing fractions were pooled. Urea, concentrated phosphate buffer (equimolar mixture of NaH2PO4 and Na2HPO4, pH 7.0 at 25 ° C) and SDS were then added to final concentrations of 8 M, 240 mM, and 0.1% (w/v), respectively. The sample was then applied to a column of

326

H A P (Bio-Gel HTP) allowing 2 g H A P per m g D N A . The H A P was prepared by numerous decantings with 50 m M phosphate buffer (pH 6.8) to remove the "fines", poured into a 4 X 25 c m column, and equilibrated with additional 50 m M phosphate buffer (500 ml per 30 g HAP). After loading the sample, the column was washed with the urea-phosphate-SDS buffer (pH 7.0) until the absorbance of the eluate at 260 n m (A260) was reduced to zero. Urea was then washed from the column with 14 m M phosphate buffer, p H 7.0 (at 25 ° C) and the D N A eluted with a linear gradient from 200 to 500 m M phosphate buffer (pH 6.8). 5 to 7 ml fractions were collected. Absorbance was determined at 230, 260 and 280 n m for each fraction, and those with good spectral qualities were pooled. The pool was dialyzed against 20 m M sodium acetate, frozen, and concentrated by lyophilization. W e have also modified the above procedure to shorten the time of preparation by including a single enzyme digestion and ethanol precipitation step to replace the time-consuming aquacide concentration technique. In this case, the extraction medium contained 10 m M Na2EDTA, 500 m M NaCI, 100 m M Tris, p H 8.4 (at 25 ° C) and 1% (w/v) SDS. Lyophilized tissue was combined with preheated extraction buffer, homogenized and held at 60 ° C for 30 min as above. The homogenate was cooled to 37°C, 2 ml pronase (Calbiochem, B grade; 10 mg/ml were added for earh 300 ml homogenate, and the extract was incubated at 37 ° C for 1.1/2 h. The homogenate was then centrifuged for 30 min at 20 000 × g. The supernatant fraction was adjusted to 5 % (w/v) SDS and 5 M NaCIO4 and recentrifuged for 20 min at 10 000 × g. At these concentrations of SDS and NaCIO4 a raft was formed which contained the chlorophyll and most of the protein [6]. The SDS-protein raft was discarded and the nucleic acids in the underlying NaCIO4 solution precipitated with 2 volumes of ethanol at - 2 0 ° C overnight. The precipitate was collected by centrifugation at 20 000 × g for I h and dissolved in a small volume of 20 m M sodium acetate. The resulting solution was adjusted to 100 m M NaCl, 10 m M Tris, pH 7.5 (at 25 ° C) and 5 % (w/v) sucrose and then clarifiedby centrifugation at 20 000 × g for 20 min. The clarifiedsample was applied to a P 200 column, further purified by H A P chromatography, and concentrated by lyophilization as described above. All lyophilized D N A was stored at - 2 0 °.C. RESULTS AND DISCUSSION The DNA extracted by our methods had excellent UV spectral properties (260/230 = 2.4 and 260/280 = 1.85) and estimates of final yield bsJed on absorbance measurements agreed with those obtained by diphenylamine assay [5]. Thermal denaturation profiles showed sharp thermal transitions with about 27% hyperchromicity (Fig. 2). The DNA was readily iodinated in vitro and the labeled product displayed greater than 95% sensitivity to DNAase but was not digested by RNAase or pronase. Moreover, these DNA preparations have given satisfactory, reproducible results in hybridization and reassociation experiments.

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Fig. 2. Thermal denaturation prof'fles of Osmunda DNAs in 120 mM sodium phosphate buffer measured from continuous records of absorbance at 260 n m as a function of temperature. O. claytoniana (0), O. cinnamomea (~), and O. regalis (×). These DNAs were isolated by the first procedure.

Final yields varied somewhat but were generally high -- from 10 to 28 mg/25 g dry weight (ca. 100 g fresh weight). These yields may be compared to the 0.2 to 3.9 mg previously reported for some ferns [4] and the 2 to 6 mg isolated from equivalent amounts of Marchantia and spinach [8]. Several attempts to measure total DNA content in the fern leaf tissue were confounded by the presence of tannins and other contaminants in the initial lysate. Diphenylamine assays of crude extracts gave estimates of total DNA as much as 50% lower than the actual amount recovered after gel column chromatography. Therefore, we could not determine the isolation efficiency of our methods. However, since high yields of DNA were obtained from mature leaf tissue, in which nuclei constitute a small proportion of the dry weight, the isolation methods are probably efficient. Several ethanol precipitations are often utilized during the isolation of DNA. However, precipitation of Osmunda extracts obtained in traditional extraction media always yielded preparations which were resistant to further purification. Green [4] found that DIECA appeared to prevent the formation of strong bonds between polymerized phenolics and DNA in extracts from some fern

328 species, but the presence of DIECA in extracts of Osmunda did n o t prevent brown-hued ethanol precipitates. Gel chromatography a n d / o r HAP chromatography in urea-phosphate buffer were n o t sufficient to purify the DNA from these precipitates although these same procedures were successful with the non-precipitated crude extracts. However, the ethanol precipitates formed from pronase-treated extracts in the presence of 5 M NaC104, although also brown, were easily purified by t h e techniques described. In summary, our extraction procedures combine the techniques of perchlorate-ethanol precipitation [6], gel c h r o m a t o g r a p h y [9,10], and gradient elution from HAP [11]. None of these approaches alone was successful with the Osmundas. We believe t h a t this combination o f procedures can be applied to a wide variety o f plant species where secondary plant products have made DNA extraction difficult. ACKNOWLEDGEMENTS This research has been supported by a National Science F o u n d a t i o n Grant (GB 38242), a University o f Massachusetts Faculty Research Grant to W.F. Thompson, and funds from the Carnegie I n s t i t u t i o n of Washington, Departm e n t o f Plant Biology. This report is based on a portion o f the dissertation submitted by D.B. Stein in partial fulfillment o f the Ph.D. requirements at t h e University o f Massachusetts, Amherst. REFERENCES

1 J.W. Anderson, Phytochemistry, 7 (1968) 1973. 2 H. Stem, Isolation and Purification of Plant Nucleic Acids from Whole Tissues and from Isolated Nuclei, in L. Grossman and K. Moldave (Eds.), Methods in Enzymology, Vol. XH B, Academic Press, New York, 1968, p. 100. 3 L.V. Gusta and V.C. Runeckles, Can. J. Bot., 50 (1972) 1085. 4 B.R. Green, Biochim. Biophys. Acta, 254 (1971) 402. 5 K. Burton, Biochem. J., 62 (1956) 315. 6 J. Wflcockson, Biochem. J., 135 (1973) 559. 7 G. Guinn, Plant Physiol., 41 (1966) 689. 8 J.P. Laulhere and C. Rozier, Plant Sci. Lett., 6 (1976) 237. 9 R.F. Heyn, A.K. Hermans and R.A. Schilperoort, Plant Sci. Lett., 2 (1974) 73. 10 P.F. Lurquin, G. Tshitenge, G. Delaunoit and L. Ledoux, Anal. Biochem., 65 (1975) 1. 11 R.J. Britten, D.E. Graham and B.R. Neufald, Analysis of Repeating DNA Sequenc~ by Reassociation, in L. Gromunan and K. Moldave (Eds.), Methods in Enzymology, Vol. XXIX, B, Academic Press, New York, 1974, p. 363.