A method for the preparation of mammalian deoxyribonucleic acid

419

SHORT COMMUNICATIONS

BBA 93110

A method for the preparation of mammalian deoxyribonucleic acid Although the methods for the preparation of mammalian DNA are generally considered adequate at present, there are some problems and limitations. Most procedures have been designed for the extraction of DNA only from the thymus gland, which is exceedingly rich in nuclear material. It has been our experience, and that of others1, 2, that it is most difficult to prepare highly intact DNA consistently by the current standard methods from tissues other than thymus. The major work in this laboratory has, for several years, been the study of some nongenetic physiological activities associated with mammalian DNA ~. It was important to us to develop a more reproducible method of systematically preparing DNA from m a n y mammalian sources. We used as a basic starting point the extractability of DNA-histone by high ionic strength salt from the nuclear fraction*. Many variables were then studied. At one point, in order to lyse some remaining erythrocytes, a small amount of sodium deoxycholate was added to the tissue homogenization and wash fluids. Quite unexpectedly, it was immediately noted that all the subsequent steps proceeded more efficiently after addition of the minute quantity of deoxycholate. Moreover, DNA of reproducible high quality was then consistently obtained even from difficult tissues such as liver. With the knowledge that deoxycholate has been widely used in m a n y biological methods and in DNA preparations, this phenomenon was further studied. A method was developed which, aside from the deoxycholate, is similar in approach to some current procedures 1. We have successfully used it to prepare mammalian DNA for the past three years and it appears worth reporting. All procedures were performed in the cold in an ice bath, cold room or refrigerated centrifuge. The mammalian tissues were frozen in solid CO2 (--60 °) immediately after removal from the organism and stored in this frozen form. The frozen tissue was weighed and then thawed slowly (30 rain) in a volume of 6 ml/g tissue of 0.04 ~o sodium d e o x y c h o l a t e + o . I o M NaCl+o.o5 M sodium citrate (pH 7) in an ice-bath. When working with tissues containing much fibrous connective tissue (thymus, spleen, lung) it was necessary to treat the mixture in a Waring Blendor or Virtis mixer for lO-15 sec. The suspension was then gently ground at o ° in a glass-teflon homogenizer. It was homogenized just sufficiently to break all the cells and no more. The extent of homogenization required for each type of tissue studied was determined by microscopic examination and cell counts. The suspension was then centrifuged at 3000 rev./min for IO min. The supernatant was poured off and the sedimented "nuclear" fraction washed with the same initial volume of the ice-cold 0.04 o//.osodium deoxycholate-saline-eitrate. This washing of the sediment was repeated four times; that is, until the supernatant wash fluid was clear. (The supernatant wash fluid should not become even slightly viscous; if some viscosity does develop during the repeated washings, it means that the concentration of deoxycholate is too high and the insoluble DNA-histone is dissoeiating~.) 13iochim. Biophys. Acta, 114 (1966) 419-422

420

SHORT COMMUNICATIONS

The final sediment was then extracted vigorously with twice the initial volume of ice-cold 2 M NaC1 for 3 min at o °. The mixture was centrifuged at 4ooo rev./min for 15 min. The supernatant containing the extracted DNA plus protein, was poured into a flask; an equal volume of chloroform-amyl alcohol (IO : I) was added; the mixture was shaken thoroughly and centrifuged. A total of lO-12 of these deproteinization steps was performed on each DNA extract. The final aqueous DNA supernatant was carefully separated from the chloroform phase and filtered. Two volumes of ethanol were then rapidly added to the cold 2 M N a C I + D N A solution. The precipitated white DNA fibers were gathered by winding on a glass rod and gently pressing the excess fluid out on the side of the beaker. The fibers were dissolved in NaCl solutions of varying concentrations and in standard buffers (pH 7) with shaking (3o-6o min) necessary. These moderately viscous, clear solutions were filtered and stored in aliquots frozen at --2o ° or --6o °. The final concentrations of DNA in these solutions ranged from IOO #g/ml to 3oo #g/ml. Using the method described above, it was decided to concentrate the work on DNA prepared from three different sources: I, rabbit liver (metabolically active, non-dividing cells with a low concentration of DNA); 2, calf thymus (many mitotic cells with a high concentration of DNA); 3, Ehrlich ascites tumor cells. During a period of three years some 14 DNA preparations were made from these three sources for use in other studies in progress in this laboratory. Table I shows some physical TABLE I PROPERTIES

OF

THREE

TYPICAL

DNA

Re/erence

N/P P r o t e i n c o n t e n t (%) R N A c o n t e n t (°/o) e(p), 259 m l , AA259m~[-d g31ln]~ zsgm/z/A zs0m/Z M e l t i n g t e m p e r a t u r e (°C) °/o H y p e r c h r o m i c i t y , i o o ° I n t r i n s i c v i s c o s i t y ' * (dl/g) B a s e a n a l y s i s (%) Adenine Guanine Thymine Cytosine Guanine + cytosine

5,6 7 8

9,1o 9,IO

PREPARATIONS

Call thymus

FROM

MAMMALIAN

Rabbit liver

TISSUES

Ehrlich ascites cells

Range o[ all preparations" 1.59-1.72 < I
< I <0. 5 6OlO 2.21 1.89 87. 5 4° lOl

1.68 < I <0. 5 586o 2.32 1.86 87 40 75

< I <0. 5 614o 2.17 1.88 87.8 39 89

3o-3 22.5 26.8 2o.5 43

29.8 21-7 28. i 2o.4 42

3o.6 21.5 27. 4 2o.5 42

12

" R e p r e s e n t s 6 D N A p r e p a r a t i o n s f r o m calf t h y m u s , 4 f r o m r a b b i t liver, a n d 4 f r o m E h r l i c h a s c i t e s cells. ** V i s c o s i t y w a s m e a s u r e d in a m u l t i g r a d i e n t capillary v i s c o m e t e r 11 s u p p l i e d b y t h e C a n n o n I n s t r u m e n t Co. (State College, Pa., U.S.A.). T h e i n t r i n s i c viscosities were d e t e r m i n e d b y e x t r a p o l a t i o n to zero c o n c e n t r a t i o n a n d zero shear.

and chemical properties of three typical individual DNA preparations. The analytical methods are noted as references in the Table. The DNA was highly purified with no demonstrable protein or RNA. The low extinction coefficients and the high intrinsic viscosities indicate that the material was in a highly polymerized, undenatured, Biochim. Biophys. Acta, I14 (1966) 419-422

SHORT COMMUNICATIONS

421

double-stranded form. An estimate of the range of approximate molecular weights, obtained from the viscosity data 13, was from 7-11" lO6. The yields ranged from IO ~o to 30 % of the nucleic acid in the starting material. Most important was that by using this method there were no differences whatsoever between the DNA solutions prepared from the different types of mammalian tissues. The particular innovation introduced is the presence of a small quantity of deoxycholate (0.04 %) in the solutions used to homogenize the tissue and to wash the nuclear sediment repeatedly. Although deoxycholate is an important ingredient in many biological preparatory methods, including extraction of DNA from bacteria, it has not been previously utilized for the preparation of mammalian DNA in exactly the manner described here. The exposure of the nuclear material to the deoxycholate probably results in the breaking of important lipoprotein links in the nuclear matrix and membranes. Apparently, mild disruption of the nuclear organelle permits a more reproducible subsequent extraction of higher quality DNA. It is interesting to note that KIRBY,working with an entirely different set of observations and with the phenol procedure, has come to some similar conclusions. In a recent review 14 he has suggested that the chromosomes and the DNA of multicellular organisms are covered by lipoproteins. Furthermore, he has indicated the necessity of using lipophilic anions and chelating agents to break the chromosomal lipoprotein to adequately extract the DNA by his phenol method. It was found that our preparatory method was significantly more effective when frozen-and-thawed tissues were used as the starting material rather than fresh tissues. This has been noted occasionally before by other investigators and, indeed, KAY,SIMMONS AND DOUNCE15 have stated in no uncertain terms that their methods are applicable only to frozen tissues. The explanation of this phenomenon may be similar to the role of deoxycholate. It is likely that the freezing and thawing breaks bonds in the lipoprotein membranes and matrix of the cell nuclei permitting easier extraction of high quality DNA. In summary, a method has been described, using small amounts of deoxycholate, which results in the reproducible preparation of highly intact DNA from a variety of mammalian tissues. We wish to thank Dr. J. OPPENHEIMER and his laboratory for assistance with the protein determinations. These experiments were performed during the tenure of an Established Investigatorship of the American Heart Association (J.P.S.) and were supported by grant AM-o2286 from the National Institutes of Health.

Lucy and Henry Moses Research Laboratories, Medical Division Monte[lore Hospital and Medical Center, New York, N.Y. (U.S.A.)

J. PHILIP SAVITSK¥ FRIEDA STAND

i E. CHARGAFF, in E. CHARGAFF AND J. ~N~. DAVIDSON, The Nucleic Acids, Vol. I, Academic Press, N e w York, 1955, p. 307 • 2 S. ZAMENHOF, in S. P. COLOWICKAND N. O. KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, N e w York, 1957, p. 696. 3 J- P. SAVITSKY, Am. J. Physiol., 203 (1962) 929. 4 A. E. MIRSKY AND A. W. POLLISTER, J. Gen. Physiol., 3 ° (1946) 117. 5 T. S. MA AND G. ZUAZAGA, Ind. Eng. Chem., 14 (1942) 280. 6 B. I. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375.

Biochim. Biophys. Acta, 114 (1966) 419-422

422

SHORT COMMUNICATIONS

7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (I951 ) 265. 8 J. p. SAVITSKY AND V. STAND, Nature, 207 (1965) 758. 9 J. MARMUR AND P. DOTY, Nature, 188 (1959) 1427. io J. MARMUR AND P. DOTY, J. Mol. Biol., 5 (1962) lO9. I I j. EIGNER, Doctoral Thesis, H a r v a r d Univ., April, 196o. 12 A. BENDICH, in S. P. COLOWICK AND N. 0 . KAPLAN, Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p- 715. 13 P. DOTY, ]3. ]3. McGILL AND S. A. RICE, Proc. Natl. Acad. Sci. U.S., 44 (1958) 432. 14 K. S. KIRBY, in J. N. DAVIDSON AND W. E. COHN, Progress in Nucleic Acid Research and 2VIolecular Biology, Vol. 3, Academic Press, New York, 1964, p. I. 15 E. R. M. KAY, ~NT.S. SIMMONS AND A. L. DOUNCE, J . A~/~. Chem Soc., 74 (1952) 1724.

Received September 2nd, 1965 Biochim. Biophys. Acta, 114 (1966) 419--422

BBA 93113

The chromatographic separation of ribonucleic acids DEAE-cellulose, ECTEOLA-cellulose and methylated serum albumin on kieselguhr have been widely used for the separation of rRNA from sRNA 1-*. It is, however, difficult to recover rRNA from the first two materials without degradation, and columns of methylated serum albumin on kieselguhr have only low capacity. Nucleic acids interact with DEAE-cellulose and ECTEOLA-cellulose with nonionic as well as ionic forcesS; this communication describes an attempt to make use of these non-ionic forces for the chromatographic separation of rRNA from sRNA. The addition of ethanol to a nucleic acid solution increases the chemical activity of the nucleic acid 6, and a sufficient concentration of ethanol should enable non-ionic sorption of nucleic acid to occur on unmodified cellulose. Preliminary work showed that in o.I M NaC1 sorption of yeast rRNA, prepared according to CRESTFIELD, SMITH AND ALLEN 7, and yeast sRNA, prepared according to HOLLEY et al. 8, occurred when the ethanol concentration was greater than 2 1 % and 30 %, respectively. In 0.59 M NaC1 the required ethanol concentrations were 8 °/o for rRNA and 20 °/'0 for sRNA. In the column chromatographic separations Whatman cellulose powder CF I i was used. The cellulose was suspended in distilled water, loaded into a column (18 c m × 2 . 2 cm) and washed with distilled water and with o.I M NaCl-ethanol (65"35, v/v). A mixture of rRNA and [14Clisoleucine-labelled sRNA (labelled according to HELE AND FINCH9) in o.I M NaCl-ethanol (65:35, v/v) was applied to the column and eluted first with a solvent of the same composition and then with distilled water. It was shown in a separate experiment that the radioactivity is distributed evenly throughout the sRNA when it is eluted from the column under the same conditions, thus the I14C]isoleucine label is a legitimate method for the measurement of sRNA in the presence of rRNA in this experiment. The elution histogram is shown in Fig. I and the concentration of the radioactivity in the first peak shows that the method gives a good separation. The recovery of both nucleic acids is in the region of Ioo o/ /0" Biochim. Biophys. Acta, 114 (1966) 422-424