ANALYTICAL

BIOCHEMiSTRY

Purification Removal

65, 293497

of DNA

f 1975)

by Affinity

of Polysaccharide

Chromatography: Contaminants

MARVIN EDELMAN Department

ofPlant

Genetics. The Weizmunn Insfifute of Science Rehovot, Israel

Received September 25, 1974; accepted November

19, 1974

DNA, as isolated, is often contaminated with polysaccharidelike components. To overcome this difficulty a simple and rapid procedure has been developed based on the principle of affinity chromatography. Deproteinized DNA fractions are introduced onto a column of concanavalin A linked to Sepharose. Glycogen and starchlike polysa~~haride contaminants are effectively bound to the carbohydrate exchange resin while nucleic acids pass through (recovery, >9.5%). The column can then be regenerated for further use. DNA samples from a variety of organisms have been successfully freed of polysaccharide contaminants by this method.

The DNA purification procedures commonly employed (1,2) for bacterial, plant and animal cells are generally effective in removing lipids, proteins and RNA, but are less satisfactory in the case of polysaccharides (3). The latter often follow DNA tenatiously through the various purification steps, including chloroform or phenol extractions and alcohol precipitations. Enzymes such as alpha amylase, which specifically hydrolyze polysaccharides, cannot be effectively used since they are usually contaminated with nucleases. In fact, commercial preparations of alpha amylase serve as a convenient source for obtaining purified nucleases (4). Removal of polysaccharide contaminants by preparative CsCl density gradient cent~fugation is also largely ineffectual since many polysaccharides have buoyant densities similar to those of DNA (3). This is quite a widespread phenomenon, affecting many biological groups (see Table 1). In most of the cases shown in table 1 the polysaccharides could be degraded by alpha amylase, pointing to a common glycogen or starchlike composition among them. With this in mind, experiments were initiated with various carbohydrate-exchange resins in an attempt to selectively trap the polysaccharide contaminants by means of affinity chromatography. This paper presents results using columns of concanavalin A (Con A) linked to Sepharose. With this resin, DNAs from a variety of sources were successfully freed of their polysaccharide contaminants. 293 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

294

MARVIN

EDELMAN

TABLE BUOYANT

1

DENSITIES OF SELECTED DNAs AND THEIR CONTAMINATING POLYSACCHARIDES

Density in CsCl (g/cm3)” Biological

group

Bacteria Blue-green algae Protozoa Fungi Insects Mammals

Organism Eschericia co/i* Nod&aria sphaerocarpa Tetrahymenapyriformis Aspergillus nidulans Spodoptera littoralis

Mouse

DNA

Polysaccharide

1.710 1.698 1.686C

1.711, 1.689 1.698 1.700. 1.691

I .667 1,729, 1.704 1.674’ 1.675 1.681 1.70-1.67

Reference 5 6 7 8 9 10, 3

a The approximate overall density range reported for DNAs is 1.750-1.680 g/cm3 (11) and for polysaccharides, 1.7.50-1.650 g/cm3 (6). b Cell lysate. c Calculated from data in Ref. 7.

MATERIALS AND METHODS

Con A linked to agarose is available commercially (Miles Laboratories, Kaukakee, IL; Pharmacia Fine Chemicals, Uppsala, Sweden). Alternatively, the carbohydrate-exchange resin can be prepared from Con A and Sepharose 4B (12,13). Glycosylex A, the commercial resin from Miles Laboratories, was used in this study. One ml columns, equilibrated with 20 vol of 0.15 M NaCl, 0.015 M sodium citrate, pH 7.3 (SSC), were prepared in Pasteur pipettes. After elution of the nucleic acids, bond polysaccharides were released from the column by washing with 5-10 vol of 5% cY-methylmannoside (14) (methyl-a-D-mannopyranoside; Pfanstiche Laboratories, Waukegan, IL). The column was later regenerated by washing with several volumes of buffer. CsCl-density gradient centrifugation and detection of polysaccharide bands was carried out as previously described (6). RESULTS AND

DISCUSSION

DNA extracts from mouse liver often contain variable amounts of glycogen. An extract of such material (5.0 AZ&, purified by the method of Marmur (l), was loaded onto a Con A-Sepharose column and fractions collected. About 90% of the A,,, was eluted in the first 2 ml after the void volume and a further 8% in the next 3 ml: total recovery of A,,, was > 98%. A simple test is available for determining polysaccharide contamination of DNA samples centrifuged to equilibrium in CsCl(6,7). Accordingly, the analytical ultracentrifugation patterns of material before and after passage through the carbohydrate-exchange resin were compared. Fig. 1 shows the uv absorbance and Schlieren refractive

PURIFICATION

OF

295

DNA

BEFORE COLUMN 1.731 1.691 ! I.7001 1.660 I II 1

AFTER COLUMN I.731

1.691

uv

absorbance

Density(g/cm31 FIG. 1. W&density gradient patterns of mouse liver DNA extract before and after column chromatography. About 0.3 A,,, units from loaded and eluted samples were centrifuged to equilibrium in 5 M CsC1 at 44,770 and 25°C in an analytical ultracentrifuge (16). UltravjoIet-absorbance and Schlieren refractive index patterns were photographed after 20 DNA): I.700 gicm3, mouse mainband hs. 1.731 g/c&, density standard (M. lysodeikticus DNA; 1.691 g/cm”, mouse satellite DNA; 1.680 g/cm3, polysaccharide band (glycogen).

index patterns. Notice the absence of the band at 1.680 g/cm3 from the DNA sample &ted from the column. This band can be identified as a polysaccharide (glycogen) on the basis of its low specific absorption (compare refractive index with uv absorbance) which indicates that it is scattering rather than absorbing light (6). The material at 1.680 g/cm3 also proved to be insensitive to deoxyribonuclease but sensitive to a! amylase. A more complete picture of the working of the column is shown in Fig. 2. In this case DNA extracted from the filamentous fungus Tric~~der~a viride was chromatographed. After elution of all unbound material absorbing at 260 nm with saline-citrate buffer, affinity-bound material (having optical density at 320 nm due to light scattering) was eluted with 5% cr-methylmannoside. Peak fractions from each eluate were then analysed in the ultracentrifuge, as was the unfractionated DNA extract loaded on the column. The refractive index patterns at the bottom of Fig. 2 clearly distinguish between the DNA peak (1.713 g/cm3) eluted with SSC in fraction 3 and the starchlike polysaccharide peak (1.671 g/cm3) eluted with cr-methylmannoside in fraction 13. The data show that within the limits of detection of the analytical ultracentrifuge, the two components have been completely separated from one another.

296

MARVIN

EDELMAN

FRACTION

NUMBER

(I-METWLMANNOSIOE

FRACTLON

13

DENSITYtqkm’)

Fro. 2. Chromatography and analysis of Tn’chodevma DNA extract. Thirty-five A, units of DNA extract in 1 ml of SSC were loaded on a column of Con A-Sepharose. The flow rate on the column was 1 mh’min. Fractions l-10 were eluted with SSC. After fraction 11, the elution buffer was changed to 5% alpha Methylmannoside. One ml fractions were collected throughout. A,,,,, @---a; A,,,, 0-O. The amount of polysaccharide eluted by the methyl mannoside was estimated to be 1.5 mg equivalents of glucose based upon its spectrophotometric scattering profile (6). Analytical ultracentrifugation in CsCl is described in Fig. 1. I.731 g/cm”, density standard; 1.713 g/cm3, ~r~choder~a DNA (sensitive to DNase); 1.671 g/cm”, polysac~haride band (sensitive to alpha amylase).

Similar experiments have been performed with DNA extracts from higher plants, insect larvae and blue green algae. In each case DNA was recovered from the column in high yield and free from detectable polysaccharide bands. The thermal denaturation properties and szOvalues of the eluted DNAs were comparable to those of the nonchromato~aphed material. RNA extracts from fungi and higher plants have also been purified by this method. Finally, it should be emphasized that Con A will not indescriminately bind all polysaccharide contaminants. The specificity of this lectin is particularly for carbohydrates containing glucose, fructose or mannose as terminal groups (14,15). Its general applicability for nucleic acid purification can probably be traced to the fact that a substantial variety of plant and animal nucleic acid fractions contain starchlike or glycogenlike polysaccharide contaminants which are strongly bound by Con A.

PURIFKCATION

OF DNA

297

ACKNOWLEDGMENTS The expert technical assistance of Mrs. Dina Heller and Mr. Leon Esterman is gratefully acknowledged.

REFERENCES 1. Marmur, J. (1963) Methods Enzymol. 6, 726-738. 2. Kirby, K. S. (1968) Methods Enzymol. 12B, 87-99. 3. Segovia, Z. M. M.. Sokol, F., Graves, 1. L.. and Ackerman% Bi#phy~,

W. W. (1965) Biockim.

Acta

9.5, 329-340. (1973) Eur. J. Biochem.

Vogt, V. M. 33, 192-200. 5. Schumaker, V. N., and Wagnild, J. (1965) Biophys. J. 5, 947-964. 6. Edelman, M., Swinton, D., Schiff, J. A., Epstein, H. T., and Zeldin, 3 (1967) Bac-

4.

teriol. 7.

8. 9. 10. 1 I. 12. 13.

14.

Rev.

31, 315-331.

Brunk, C. F., and Hanawalt, P. C. (1966) Exp. Cell Rrs. 42, 406-408. Edelman, M., Verma, I. M., and Littauer, U. Z. (1970) f. Mol. Hoi. 49, 67-83. Kisiev, N., Edelman, M., and Harpaz, I. (1971) J. (nr,ert. fathom. 17, 199-202. Counts, W. B., and Fiamm, W. G. (1966) Biochinz. Biophys. Acta 114, 628-630. Sober. H. A., ed. (1970) Handbook of Biochemistry, Selected Data for Molecular Biology, 2nd edition, pp. H I3- 1.5,Chemical Rubber Co., Cleveland. Aspberg, K., and Porath, J. (1970) Acta Chem. Scand. 24, 1839-1841. Lloyd, K. 0. (1970) Arch. Biochem. Biophys. 137,460-468. Goldstein, I. J., Hollerman, C. E., and Merrick, J. M. (1965) Biochim. 3i~p~zys. Acta 97, 68-76.

15. Goldstein, I. J., and So. L. L. (1965) Arch. Biochem. Biophys. 111, 407-414. 16. Schildkraut, C. L., Marmur, J., and Doty, P. (1962) J. Mol. Biol. 4, 430-443.

Purification of DNA by affinity chromatography removal of polysaccharide contaminants.

ANALYTICAL BIOCHEMiSTRY Purification Removal 65, 293497 of DNA f 1975) by Affinity of Polysaccharide Chromatography: Contaminants MARVIN EDEL...
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