ANALYTICAL BIOCHEMISTRY 51, 510-515 (1973)

Micropreparative Equilibrium Density Centrifugation in the Analytical Centrifuge

JOHN DARNELL ASD JOEL ROSENBL001\1

Department of Biochemistry and Center for Oral Health Research, School of Dental Medicine, L’niversity of Pennsylvania, Philadelphia, PA 1.910.1

Since the introduction of equilibrium density gradient centrifugation in 1957 by iUeselson, Stahl and Vinograd (1)) this technique has been used extensively to analyze DNA and RNA preparations. The most com- monly used salt has been CsCl and the exact bouyant density of a particular DNA in concentrated salt, solutions has proven to be a sensi- tive function of the base composition of the DNA (2), as well as of other factors (2-5). The separation of DNA species by density has led to preparative equilibrium sedimentation fractionation techniques. These preparative methods have used either swinging buckets (6) or fixed angle rotors (7) and centrifugation for 48-72 hours in order to achieve equilibrium. Fractionation of the contents of the tube has then been achieved either by puncturing the bottom of the tube and collecting drops or by any one of a number of commerically available apparatus. The fractions thus obtained could then be analyzed for the presence of nucleic acid by spectrophotometry, radioactive labeling or other pro- cedures. Because of the dimensions of the tubes involved 3.5-5 ml of the CsCl solution are necessary and for optical observation of the DNA at the end of the run at least 3 pg of DNA are required, In addition, the progress of the run can not be monit,ored in the preparative experiments.

Where only small amounts of DNA are available (< 1 pg) a prepara- tive method using the analytical ultracentrifuge would be advantageous. In addition the progress of the run can be monitored and the time of centrifugation is considerably shortened. There have been no reports in the literature of attempts to fractionate the contents of analytical cells after equilibrium centrifugation. In the present communication we re- port a successful method for achieving such fractionation.

MSTERIALS AND METHODS

Preparation of DNA

Micrococcus lysodeikticus was grown in brain-heart infusion contain- ing .25 ,&X/ml 14C-thymidine until stationary phase was reached. DNA

510 Copyright @ 1973 hy Academic Press. Inc. All rights of reproduction in any form reserved.

MICROPREPARATIVE CENTRIFUGATIOK 511

was t,hen extracted from 2 gm wet weight of the cells by modification of the method of Marmur (8). After the initial deproteinization with chloroform and precipitation with alcohol the DNA was resuspended in saline citrate and treated with 50 hlg/rnl ribonuclease for 30 minutes at 37”. Pronase was then added to a final concentration of 500 pg/ml and

FIG. 1. Micro preparative apparatus for recovery of contents of analytical ultra- centrifuge cell. A. Apparatus in position. A twenty-t,hree gauge stainless needle with a slightly beveled tip was attached to PE-10 tubing (Clay-Adams) for recovery of cell contents. The needle was beveled to prevent blockage of flow when it was lowered to the bottom of the cell. The needle was supported by concentric cylinders of Tygon tubing, with a layer of soft white silicone rubber tubing pro\-iding the air-tight seal against the cell housing. Air is pumped into the top of the cell though another needle which diagonally penetrates the tubing cylinders. B. Method of at- tachment to microscope barrel for smooth and precise lowering of needle into cell. C. Wedges fashioned from Epon embedding material and glued down with same is shown installed in centerpiece. View is a section through the centerpiece with windows on the left and right. The wedges provide better draining of cell contents.

512 DARNELL AND ROSENBLOOM

the solution incubated for 4 hours at 37”. The deproteinization with chloroform was then repeated, the DNA precipitated with alcohol, and then redissolved in saline citrate. The final specific activity was 140 cpmhng.

E. coli B (T-) was grown to a Klett reading of 120 in 20 ml Tris- glucose medium supplemented with 10 pg/ml thymidine, and infected with T4 bacteriophage at a multiplicity of 5 in t,he presence of 50 pg/ml t,ryptophan. After 5 minutes 50 PCi 3H-thymidine was added. Incubation was continued until the Klett reading declined and then the cells were lysed with CHCl,. The lysate was treated with 50 pg/ml deoxyribo- nuclease and 50 &ml ribonuclease for 15 minutes at 37”. Cell debris was removed by filtering through celite and centrifuging at 5,000 rpm for 15 minutes. The phage were pelleted by centrifugation at 30,000 X g for 1 hour and then resuspended in .05 M Tris buffer pH 7.5. DNA was prepared from the phage by phenol extraction, and the DNA solution dialyzed against saline citrate (9). The specific activity was 900 cpm/pg.

Construction of Device for Removal of Contents from Analytical Cells and Modification of Analytical Cell Bottom

Figure 1 provides a detailed diagram and description of the fractiona- tion device which was constructed from readily available materials. Basically the device consists of a needle attached to a rack and pinion for smooth insertion into the cell. Concentric cylinders of Tygon tubing (and silicone rubber) provide (i) an air-tight seal at the opening in the metal housing, (ii) an entrance for air into the top of the cell, (iii) an exit for small bore tubing at.tached to the needle. The volume in this piece of tubing is only 6 ,J so there is very little opportunity for mixing. Flow from the cell is cont,rolled by pumping air into the top of the cell with the use of a syringe pump of the type used for delivery of samples into a gas chromatography apparatus. The drop size is about 8 ~1. Because the analytical cells have a flat bottom some puddling of liquid tended to occur which distorted the bands. The cells were therefore modified as indicated in Fig. 1 by gluing small wedges on epon to the bottoms. Com- plete drainage of the cells was achieved in this way.

Termination of Centrifuge Run and Filling of Cells

After equilibrium has been reached, the drive is turned off as usual but when the speed has decreased to 5,000 rpm the vacuum pump is turned off and air is introdued into the chamber. The chamber is opened when the rotor is at 2,000 rpm so that the gradient is stabilized against vibra- tion from the chamber shielding lift’. The rotor is disconnected from the drive with a minimum amount of shaking and the cell gently removed

MICROPREPARATIVE CENTRIFUGATIOK 513

from the rotor and immediately rotated 90” so that the density gradient is vertically aligned with gravity. We have found that the minimum amount of mixing occurs when the cell is maximally filled, with either a small or no air bubble being present. Experiments with two cell opera- tion can be performed.

RESULTS AND DISCUSSION

Figure 2 represents the refractive index and corresponding density of fractions recovered from a modified 4” sector cell. The plot is linear over about 70% of the central portion of the recovered gradient with some distort,ion at the bottom and top. The bottom distortion is probably caused by the wedges, while that at the top may be due to surface tension effects as the meniscus descends during recovery of the gradient. Figure 3 is a densitometer tracing of a photograph ‘taken with the ultra- violet optical system of an equilibrium run containing a mixture of 1 pg T4 and 4.8 pg Micrococcus DNA. The two DNA’s are well resolved. Figure 4 illustrates the recovery of the radioact’ive DNA from the cell from this same experiment. Resolution is complete. The apparent. rela- tive amounts of the two types of DNA is, of course, different from that observed in the photographs since the specific activities are different. The positions of the recovered radioact’ive peaks in Fig. 4 transposed to the scale of Fig. 1 are indicated in Fig. 1 along with the range of values found in the literature for these two DNA’s The position of the Micro- coccus DNA is well within the literature range, while t’hat for T4 DNA is

’ 3y60w Fraction Number

FIG. 2. Plot of the refractive index and corresponding density of fractions re- covered from density gradient. Four drop fractions were collected directly onto the prism of a Bausch & Lomb AbbC refractometer. Arrows indicate recovered positions of M. Egs. and T4 DNA from the experiment described in Figs. 3 and 4. Solid bars next to the line indicate the range of values found in the literature for these two DNA’s,

514 DARNELL AND ROSENBLOOM

FIG. 3. Densitometer tracing of photograph of an equilibrium density gradient run. One pg of T4 DNA (900 cpm) and 4.8 pg of M. lysodeikticus DNA (673 cpm) were centrifuged to equilibrium in a CsCl solution in standard saline citrate (start- ing refractive index 1.4005) in a modified 4” sector cell. The photograph was taken with 265 rnp light (10) after equilibrium had been reached (24 hours) at 44,0@3 rpm at 25”. Unlabeled arrow indicates unmodified cell bottom. Although the installed wedges mask the bottom part of the cell they only displace a volume of 25 ~1 in a total volume of 750 ~1.

FRACTION NUMBER

FIG. 4. Recovery of radioactive DNA from cell. The conditions of the centrifuge run are de&bed in Fig. 3. Two drop fractions were collected directly into counting vials. Six-tenths ml water and 4.5 ml of a Triton baaed counting solution (11) was added and the fractions counted in a scintillation counter by standard double labeling techniques. T4, (0. .O..O) ; M. ZIG. (O-0-0).

XICROPREPARATIVE CEKTRIFCGATIOW 515

near the top of the values. Reasonable estimates can then be made for the bouyant density of an unknown DNA from its position in the gradient. However, because of the small volumes involved, it is not practical to collect fractions both for refractometric measurements and radioactivity from the same gradient. A better procedure is to use a marker DNA of known density for int’ernal calibration. Similar results to those presented have been obtained using 2” sector centerpieces, but the problems involved at’ the bottom and top of the gradient affect a larger fraction of the recovered volume, and the use of 4” sector cells is recommended. This technique should prove useful for experiments in which the amount of nucleic acid is limiting since as little as .3 pg can easily be detected optically and only a few hundred counts are required.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health research grants AM- 14439 and A1M-14526, National Institute of Dental Research grant DEa2623, and the American Heart Association grant 70717. The authors would like to thank Mr. John McKitrick for preparing the Micrococcus DNA and Mr. John Merlie for preparing the T4 DNA.

REFERESCES

1. MESELSON, M., STAHL, F. W.: .4x11 VIXOGRAD, J., Proc. N&l. Acnd. Ski. 43., 581

(1957). 2. SCHILDKRAUT, C. L.. MARML-R, J., AND DOTY, P., J. Mol. Biol. 4, 430 (1962). 3. HEARST, J. E., IFFT, J. B., AND VINOGR~D, J., hoc. h’atl. Acad. hi. 47, 1015 (1961). 4. HEARST, J. E., AND VIXOGRAD. J., Proc. Natl. Acad. Sci. 47, 1005 (1961). 5. ERIKSON. R. L., AND SZYBALSKI, Iv.‘.. I’irolo~2/ 22, 112 (1964). 6. KAPLAN, A. S., in “Fundamental Techniques in Virology,” Habel, K. and Salz-

man, S. P.. (eds.), Academic Press, New York, 1969. 7. FISHER, IV. D., CHINE, G. B.. AND ANDERSON. r\‘. G., Anal. Biochem. 9, 477

(1964). 8. MARIMI.R. J., J. Mol. Biol. 3, 208 (1961). 9. MANDELL: J. AND HERSHET, A. D.: Anal. Biochem. 1, 66 (1960).

10. ROSENBLOOM, J., Anal. Biochem. 19, 6 (1967). 11. TURNER, J. C., Internatl. J. Appl. Radiation Isotopes 19, 557 (1968).