Polyacrylamide gel electrophoresis of DNA

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RIOCHEMISlIlY

Polyacrylamide

Gel

(1072)

Electrophoresis

of

DNA

Following a report (I) on the separation of DNA components by disc electrophoresis in polyacrylamide gel, it was decided to attempt to fractionate the DNA of rat tumors by this method and to follow the changes in the DNA subsequent to treatment with the alkylating drug melfalan. The total DNA content of cells treated with alkylating agents has been shown to increase (2-4). The aim was to develop a method by which this replication could be studied qualitatively. The experiment,s mere carried out, using the DNA from t’wo experiment,al rat tumors--the Yoshida sarcoma and the Walker carcinosarcoma, both in ascitic form. Two sublines of each are under investigation, one sensitive and one resistant to alkylating agents. DNA was extracted by a modification of the Kirby method (5). The tumor cells were washed in 0.370 saline several times t,o lyse blood cells. They were then shaken with an ice-cold solution of 6% 4-aminosalicylic acid (sodium salt) (w/v) containing 1 gm triieopropylnaphthalenesulfonic acid (sodium salt) and 6 ml set-butanol per 100 ml (PAS) at, a eoncentration of 15 ml/gm cells, and after shaking were left t,o stand at 0” for 10 min. An equal volume of phenol reagent was added (500 gm phenol, 55 ml water, 70 ml m-cresol, and 500 mg S-hydrosyquinoline), and the mixture shaken at 20” for 1 hr. It, was then spun in a ewingout. centrifuge for 15 min at 5006 rpm, and the aqueous layer removed by suction. To the water layer was added an equal volume of PL48 solution saturated with sodium benzoate (approximately 40% w/v). Then 0.1 vol 2-but,oxyethanol was added slowly, with continuous stirring, and the DNA threads were collected on a glass rod. The DKA was washed several times with ethanol and ether, and allowed to dry at room temperature. To obtain labeled DNA samples, each animal was injected intra18 hr prior to death. peritoneally with 5 /Xi 14C-thymidine For electrophoresis, DNA was dissolved in 5% sucrose containing bromophenol blue marker, t’o give 30 Etg DNA in the 20 /*I samples used. It was run at 10 V/cm on 6 mm X 55 mm polyacrylamide gels, polymerized 613 @ 1972

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FIG. 1. Electrophoretic separation of DSA from rat Walker tumor (ordinate) radioactivity of gel sections, (abscissa) upper scale--R, value, lower scale-section number. The gel diagrams show two electrophoretic patterns of DNA from the resistant tumor. The upper diagram shows the four bands characteristic of both the resistant and the sensitive tumor when untreated, and the lower diagram shows the pattern given by the resistant tumor 24 hr after melfalan treatment. DNA was applied at the anodic end of the gel, and increasing section number therefore corresponds to decreasing molecular weight. The IS, values are indicated, and are related to molecular weight in Table 1. 30 gg samples of DNA in 20 ~1 5% SUWOSP were run in 6 mm X 55 mm cylindrical 2.4% polyacrylamide gels (6.9% cross-linked)

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COMMUNICATIONS

using ammonium persulfate initiat,or and TEMED (iV,N,N’,N’-tetramethylethylenediamine) catalyst (final composition: 2.4% total monomer, 6.9% cross-linked; 0.027% ammonium persulfate; 0.00077, TEMED), using sodium barbitone/diethylbarbituric acid !0.025 M and 0.005 M, respectively) buffer, pH 8.75. The gels were first allowed to equilibrate for 1 hr by running without the sample, and t’he DNA was then run until the bromophenol blue was approximately 5 mm from the end of the gel (approximately 70 min) . The gels were removed by rimming with a syringe and expelling them under water. They were fixed and stained overnight (2% pyronin Y/l% lanthanum acetate w/v in 15% acetic acid), and destained by transverse electrophorcsis in 5% acetic acid at 190 mA. Ribonuclease treatment produced no change in the band pattern obtained. Treatment. with 8 M urea did not alter the rumling time, Rf values, or pattern of bands, indicating that the DNA was denatured. From measurement of the distance of t,ravel of the bromophenol blue, the distance of travel of the anion front (taking the Rf of bromophenol blue to be 0.86 in 2.4% gel (6)) was calculated, and hence the Rf of each band. From the Rf, t’he approximat’e molecular weight was cst,imatecl using a calibrat,ion relating Rj and log molecular weight of RNA in 2.4% gel (7) (Table 1). Molecular Ii?{. MW

x106.

Weight 0.0 >5.3

0.1 3.10

of DNA 0.3 1.90

TABLE 1 Related to Its XI in 2.4yc, Polyacrylamide 0 .3 1.15

0.4 0.69

0 .5 0 40

0.6 0.24

0 7 0.G

0.X 0.09

Gel 0 .!I 0.05

0.10 O.o:!

The activity of labeled DNA fractions was determined by freezing the gels, and slicing into 1.5 mm discs. The discs from several gels were pooled, homogenized in 2 ml scintillation fluid (toluene/Triton X-100 2:1, containing 0.6% w/v butyl-PBD) and the volume made up to 15 ml. To each vial was added 0.5 gm Cab-0-Sil thixotropic agent to maintain an even distribution of gel particles. Activity was counted on a Tracerlab liquid scintillation counter. As well as showing changes in I’C-thymidine incorporation during successive periods following drug treatment, diffuse material which could using barbitone buffer, pH 5.75, at 10 V/cm, for approximately 70 min. The gels were fixed and stained in pyronin Y/lanthanum acetate and destained electrophoretically. The graphs show the activity of gel sections, illustrating incorporation of “C-thymidine in the unt,reated resistant tumor (0) and during IS24 hr after drug treatment (A). The diffuse low molecular weight material cannot be seen against the background color of the stained gel.

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COMMC~ICATIO~S

not be detected visually due to the high background retention of the stain was detected by its radioactivity. The electrophoretic patterns and corresponding radioactivity profiles of DNA from the resistant line of the Walker tumor untreated and 24 hr following melfalan treatment are shown in Fig. 1 as examples of the results obt#ained. Detailed results will be published separately. Using this method, the continuing synthesis of DNA in the resistant tumors was shown to involve a change in band pattern and increased thymidine incorporation into relatively low molecular weight DNA. The sensitive tumors showed a gradual loss of t#hedistinctive patterns. ACKNOWLEDGMENTS I thank Dr. J. T. Nodes for his help. This work was supported in part by the Science Research Council by the Cancer Research Campaign.

and in part

REFERENCES 1.

59P (1970). G., DANIELLI, G. B., AND P~cc~sr, E., Xatutx 207, 608 (1965). 3. CRATHORN, A. R., AND ROBERTS, J. J., ivature 211, 150 (1966). 4. HARRAP, K. R., AND HILL, B. T., Btit. J. Cancer 23, 210 (1969). 5. KIRBY, K. S., Biochem. J., 66, 495 (1957). 6. RICHARDS, E. B., AND CRATZER, W. B., in “Chromatographic and Electrophoretic Techniyues” (I. Smith, ed.), Vol. II, p. 419. Heinemann, London/Wiley, New York, 1968. 7. GASKILL, P., AND RABAT, D., Proc. ivat. Acad. Sci. u. s. 68, 72-75 (1971). WILLIAMSON, 2. LEVIS, 8.

R., Biochem.

J., 177,

SUSAN

Biochemistry Department, Brunei Kingston Lane Uxbridge Middlesex, England Received September 25, 1971

University,

GREGSON