Fractionation of oligodeoxynucleotides by polyacrylamide gel electrophoresis

ANALYTICAL

27, 193-204

BIOCHEMISTRY

Fractionation

(1969)

of Oligodeoxynucleotides

Polyacrylamide ELLIOT Department

Gel

ELSONl

of Biochemistry, Palo

AND

Electrophoresis

THOMAS

M. JOVIN?

School of Medicine, Alto, California 9.$.904

Received

by

Januav

Stanford

I;niversity,

2, 1968

This communication describes methods for the analytical and preparative fractionation of a mixture of homologous oligodeoxynucleotides by polyacrylamide gel electrophoresis (l-3). Mixtures of oligomers obtained by enzymic digestion of dAT, the alternating copolymer of deoxythymidylate and deoxyadenylate (4), have been studied in analytical experiments utilizing a staining procedure for detection. The oligomers d(AT), were resolved over the range from n = 3 to approximately n = 30. In preparative experiments fractionated oligomers were eluted from the gel with resolution over a range from n = 3 to about n = 23. The separation of oligodeoxynucleotides in this size range requires gels of relatively high polyacrylamide content. Factors governing resolution of the oligomers and the nature and solution of problems arising from the use of high acrylamide concentrat’ions are discussed. Characterization of the oligomers, d (AT) %,with respect to end groups, molecular weight, and helix-forming properties is being published elsewhere (5 I. MATERIALS

AND

METHODS

Reagents

Acrylamide #5521, N,W-methylenebisacrylamide (“bisacrylamide”) #8383, Ar,N,N’,N’-tetramethylenediamine (“Terned”) ,#8178, and acridine orange #1757 were obtained from Distillation Products Industries. The acrylamide was recrystallized from hot acetone and exhaustively dried in vacua. Riboflavin and toluidine blue 0 were obtained from Matheson, Coleman & Bell; 2-amino-2- (hydroxymethyl) -1,3-propanediol (“Tris”) “Trizma Base,” from Sigma Chemical Company; sucrose, from 1 Present address: York 14850. ‘Present address: West Germany.

Department

@ 1969 by

Press,

Max-Planck

of

Chemistry,

Institut

fiir 193

Academic

Inc.

Cornell physikalische

University, Chemie,

Ithaca,

NPR.

GGttingen.

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Mann Research Laboratories; glycine, from California Corporation for Biochemical Research; isobutanol, ethylenediaminetetraacetic acid (“EDTA”), and bromphenol blue, from Baker Chemical Company; and bacterial alkaline phosphatase (“BAP”) and bovine pancreatic DNase I (once crystallized), from Worthington Biochemical Company. DNA polymerase was fraction VIII (6). DEAE paper was Whatman DE-20. cu-32P-deoxythymidine triphospha.te was supplied by International Chemical and Nuclear Corporation and deoxythymidine triphosphate and deoxyadenosine triphosphate by P-L Biochemicals. All polynucleotide and oligonucleotide concentrations are expressed in terms of nucleotide phosphorus. Preparation

of dAT Oligomers

.A mixture of dAT oligomers was prepared by partial digestion of macromolecular dAT with pancreatic DNase. The macromolecular dAT was prepared with Escherichia coli DNA polymerase.3 Labeled dAT was obtained by addition of cr-32P-deoxythymidine 5Ctriphosphate to the polymerase reaction mixture. Digestions with bovine pancreatic DNase were carried out at 37” in 0.1 M Tris-HCl, pH 7.0 (25’)) and 0.01 M MgCl,. For reasons irrelevant to the present discussion the course of digestion was followed by observation of the polarization of fluorescence of acridine orange bound to the DNA (7, 8). The ratio of dye to DNA phosphate was 0.01. Measurements of the polarization of fluorescence were performed as described by Stryer (9). The fluorescence was excited by light at 504 mp. The emitted light was isolated from the exciting light by filters (Corning CS 3-67, 10% and 30% transmission at 545 and 570 rnp, respectively). The change in the polarization of fluorescence from its initial value (about 0.25) as a function of the degree of digestion was calibrated against the distribution of oligomer sizes in analytical gel experiments. For the preparative experiments the digestion was stopped when the polarization had dropped 0.06 from its initial value. A more accessible physical parameter such as viscosity should also be capable of serving as an indication of the degree of digestion in experiments of this sort. The digestion was stopped and the enzyme inactivated by making the reaction mixture 0.01 M in EDTA and 0.1 M in NaOH. After 15 min the digest was neutralized with HCl and Tris and dialyzed, first against 0.2 M NaCl, 0.1 M trisodium citrate, and then against 0.003 M trisodium citrate. For electrophoretic fractionations, the digest was made 0.1 M in sucrose ‘Since the macromolecular which had not been extensively

dAT was to be degraded, purified was satisfactory.

a preparation

of polymerase

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and bromphenol blue added as an indicator (1 ~1 and 0.2 ml of a 0.01% (w/v) solution for analytical and preparative procedures, respectively). Preparation

of Polyacrylamide

Gels

The solutions and buffers were modified from those described by Jovin stock solutions were used. Values of pH were measured at 25’ at the cited concentration. Per cent concentrations are w/v except for Temed, which is v/v. The solutions were stored at 4’C in the dark and were stable for several weeks. et al. (3). The following

Stock solution

Composition

Ll L2 L3 L4 Ul u2 u3 LB UB

60% acrylamide; 3% bisacrylamide 30/, bisacrylamide 0.15 M Tris-HCl, pH 9.0; 3yo bisacrylamide 0.003’% riboflavin; 30/, bisacrylamide 5% acrylamide; 1.5% bisacrylamide 0.23 M Tris-HCI, pH 7.2; 0.1% T6med 0.0030/, riboflavin 0.1 M Tris-HCl, pH 8.2 0.052 M Tris; 0.052 M glycine, pH 8.9

The resolving (lower) gel solution cording to the following volume ratios: Ll L2 L3 L4

(20% acrylamide)

was made ac-

0.33 0.40 0.25 0.02

Dissolved gas was removed from the solution by brief evacuation with a vacuum pump. The gel solution was poured into the column and in preparative runs the coolant was circulated to maintain the solution at the temperature at which the fractionation was to be conducted, A layer of isobutanol about 5 mm deep was placed over the solution, which was then illuminated with the fluorescent light supplied with the Buchler apparatus for one hour. After photopolymerization the isobutanol was rcmoved and the upper surface washed several times with a 1/ dilution of L3. The concentration (upper) gel solution was made according to the following volume ratios: Ul

u2 u3 Hz0

0.5 0.25 0.125 0.125

The upper surface of the resolving gel was washed twice with portions of this solution. The balance was layered over the resolving gel and cov-

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ered with a 5 mm layer of water. A 4/z hour exposure to fluorescent light was sufficient for photopolymerization. Both upper and resolving gels were opaque and white. The buffers contained in the upper (cathodic) and lower (anodic) buffer reservoirs were UB and LB, respectively. Ana.lytical

Gel Electrophoresis

Analytical gels were run at room temperature essentially as described by Jovin et al. (3) .4 Gel diameters were 5-7 mm and a current of l-3 mA/tube was used. From 0.1 to 0.3 pmole of dAT digest was applied to each gel. An upper gel 1 cm long was sufficient for concentration of the oligomer into a narrow band. After the run, the gels were stained overnight in a 1% (v/v) acetic acid solution of 0.01% (w/v) toluidine blue 0 (10). Excess dye was removed by allowing it to diffuse out of the gel into a large volume of lo/O acetic acid. For permanent records photographs or densitometer tracings were made within two or three weeks of staining since the dye slowly desorbs from the DNA. Difficulties in the use of analytical gels of high polyacrylamide concentration, similar to those described below for preparative gels, ,arose from uneven surfaces and separation of the gel from the walls of the tubes. Reduction of the bisacrylamide concentration from the 3% used in the preparative gels to 1% seemed to alleviate these difficulties partly. Preparative

Gel E1ectrophoresi.s

, Preparative gel electrophoresis was performed essentially as described by Jovin et al. (3) with the apparatus available commercially from Buchler Instruments Company.5 In this method gels are polymerized in the annular region between a cold finger and an outer cooling jacket. The cross-sectional area of the gel is 15.8 cm?. There is a small gap between the lower surface of the gel and the porous glass membrane through which ionic contact is made to the lower buffer reservoir. The material which has migrated through the lower surface of the gel is swept into a capillary in the cold finger, and thence into collection vessels by a continuous buffer flow through the gap or elution chamber. The use of gels of high acrylamide concentration presented certain difficulties, including inhomogeneous polymerization and separation of the gel from the walls of the column. More homogeneous gels were obtained by reducing the rate of polymerization. The “accelerator” N,N,N’,N’-tetramethylenediamine was omitted from the lower gel. Fur4The apparatus was constructed by Hoefer Scientific Instruments, 2609 California Street, San Francisco, California 94115. sBuchler Instruments, 1327 16th Street, Fort Lee, New Jersey 07024.

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thermore, initiation of the free radical polymerization by ammonium persulfate was replaced by photoinitiation with riboflavin. In spite of these measures there continued frequently to be some slight separation of the gel from the column walls. When this occurred, a small portion of the original mixture of oligomers migrated rapidly and without fractionation through the spaces between the gel and the wall. However, this portion was so small a fraction of the total amount of oligonucleotides that the over-all fractionation seemed to be essentially unimpaired. Another problem was presented by the lower surface of the gel. When a gel of 20% acrylamide and 0.4% bisacrylamide was run under ordinary conditions, the lower surface of the gel swelled downward and blocked the elution chamber. This was attributed to a differential hydration of the lower portion of the gel by the elution buffer which flowed past it. The difficulty was eliminated by increasing the bisacrylamide concentration to 3% (w/v) (II). The highly cross-linked gels thus produced resisted swelling and preserved a flat lower surface through several days of contact with elution buffer. Also, as indicated below, the increased crosslinking eliminated the doublet character of the pattern seen in Figure 1. Difficulties with the upper surface of the resolving gel were also encountered. According to the standard procedure (3) water is layered over the acrylamide solution in the column before polymerization to ensure a flat, smooth surface. When this was done with the 20% acrylamide solutions, however, mixing of the water with the acrylamide solution marred the developing surface. The problem was eliminated by layering isobutanol instead of water over the lower gel solution. A flat uniform upper surface was reproducibly obtained in this way. It was necessary to remove the isobut,anol immediately after polymerization in order t,o avoid dehydration of the gel (12). A certain amount of isobutanol remained in the gel; this was unimportant in the fractionation of dAT oligomers but might be disadvantageous in other applications. Finally, it was found that concentrated preparative gels could not be run at customarily high current densities. In fractionating proteins in 7.5% gels, it is usual to empfoy a current density of about 3 mA/cm2. However, when 20% gels were run at this current level, extensive deformation of the bands resulted from Joule heating. The situation was improved by using a current density of 2 mA/cm2 and this value was adopted for all experiments. Better fractionation might result with even lower current densities. Unfortunately, a long time is required for passage of oligomers of low mobility through a long gel at, low current density. The rate of migration at constant current can be increased by running the electrophoresis at higher temperatures. The convenient but arbitrary value of 15’ was selected for the fractionations described below.

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The resolving gel was 10 to 15 cm long, the concentration gel about 2 cm. The elution buffer was LB.. Elution buffer flow rates were from 0.3 to 0.6 ml per minute. In order to avoid a polarization phenomenon that increased the resistance of the system during the course of the run, the glass membrane holder was filled with tenfold concentrated LB. A pump was used to layer the sample over the upper gel in the presence of upper buffer. Due to the low ionic strength of the sample solution and the nature of the discontinuous buffer system (1) the mixture of oligomers was compressed into a very narrow band after migrating only a short distance through the upper gel. In the resolving gel, however, the dye was retarded by the gel and eventually became too diffuse to observe. Preparative runs took from three to five days. It was necessary to renew upper and lower buffers after every 24 hrs of operation. The use of the Buchler preparative polyacrylamide apparatus is well described in an instruction manual provided by the Buchler company.5 RJGWLTS

Analytical

AND

DISCUSSION

Gel Electrophoresis

Olivera, Baine, and Davidson (13) have shown that the electrophoretic mobility of DNA is relatively independent of size in free electrophoresis even for fairly small fragments. The use of gel electrophoresis in fractionating an homologous series of oligodeoxynucleotides depends on the function of the gel as a molecular sieve (14). Molecules being driven

FIG. 1. Analysis of pancreatic DNase digest of dAT in gels of varying acrylamide concentration. The bisacrylamide concentration was 0.4%. The gels were 6 cm long.

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through the gel are retarded to an extent which depends upon their size and shape and the concentration of polyacrylamide. The dependence of the resolution of dAT oligomers upon the acrylamide concentration of analytical gels is shown in Figure 1. In the gel of lowest acrylamide concentSration (10%) a substantial proportion of the oligomers migrated in a dense leading band without retardation. As the acrylamide concentration was increased, oligomers of progressively lower degree of polymerization were resolved from this mixture into discrete individual bands. Finally, at an acrylamide concentration of 20% all of the oli-

FIG. 2. High-resolution amide

was 14

and bisacrylamide cm long.

.

analysis of pancreatic concentrations were

DNase 225 rind

digest of dAT. 3%, respectively.

The

acrylThe gel

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gomers were distributed over an array of separate bands. The reason for the doublet appearance of these bands is unknown. It was necessary to enlarge the distance between the bands in order to separate them by preparative electrophoresis. This was achieved by increasing both the length of the gel and the concentration of the crosslinking compound, bisacrylamide. The latter measure abolished the doublet character of the bands and stabilized the gel mechanically as discussed above. Diffusion of the oligomers in concentrated gels occurs very slowly and does not limit the improved resolution of bands in the longer gels. Figure 2 shows an analytical gel in which higher resolution was achieved by the means described here. More than thirty bands can be distinguished. Figures 1 and 2 demonstrate the applicability of gel electrophoresis to an analytical resolution of mixtures of oligonucleotides. The ability to vary continuously the polyacrylamide content of the gel allows a convenient means of selecting for oligonucleotides in various size ranges. Application of this method to oligoribonucleotide mixtures might require a buffer system of pH lower than that used in these experiments to avoid base-catalyzed hydrolysis. During 1965-1967 methods for fractionating higher molecular weight DNA and RNA species on polyacrylamide gels were developed (15-19). The technique is therefore applicable to nucleic acids over a molecular weight range of three orders of magnitude. 2000

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Fm. 3. Elution pattern of “P-labeled dAT oligomers fractionated by preparative polyacrylamide gel electrophoresis. The gel contained 20% acrylamide, 3% bisacrylamide. The height of the resolving gel was 12 cm. The voltage varied from 200 to 300V and remained constant after the moving boundary traversed the resolving gel. Each fraction contained 5.7 ml and was collected over a 10 min period. Collection of the first fraction was begun 78 min after the oligomers passed into the resolving gel.

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200

250

300

number

FIG. 4. Elution pattern of dAT oligomere fractionated by preparative polyacrylamide gel electrophoresis. The dAT oligomers (190 Imoles) in this experiment were not labeled. The resolving gel contained 195% acrylamide and 3% bisacrylamide and was 14 cm high. The voltage varied from 200 to 350V. Fractions 27 through 193 each contained 2.2 ml and were collected over 5 min intervals. Fractions 194 through 259 each contained 4.3 ml and were collected over 10 min intervals. From fraction 260 on 8.3 ml were collected in 20 min. Fractions 233 through 293 which contained peak 20 were lost in a technical mishap. Collection of the twenty-sevent,h fraction was begun 410 min after the oligomera passed into the resolving gel.

Preparative

Gel Electrophoresis

Four preparative polyacrylamide fractionations of dAT oligomers were performed, two of 32P-labeled and two of unlabeled material. In three of these experiments more than twenty oligomer peaks were recovered. A technical mishap stopped the fourth experiment after only thirteen peaks had been recovered.” Figure 3 shows t.he elution pattern of a fractionation of 2 pmoles of 3’P-labeled dAT oligomers. Through fraction 190 there was a 60% re‘The elution pattern of the second and third preparative gels suggested that the effective acrylamide concentration was lower than its nominal value in these experiments. Mobilities of the peaks were greater and the gels less rigid than expected. This was attributed to a contamination of the acrylamide used for these experiments by acetone from which it had been recrystallized. The acetone had been rigorously removed from the acrylamide used in the fourth gel. This gel was rigid and the oligomers had low mobilities as expected. However, since the fractionation on this gel had to be halted in midcourse, it was possible to stain a portion of the gel to examine the bands that remained. They were found to be deformed as if by Joule heating in the gel. This deformation is the probable reason for the asymmetry of the eluted peaks. It suggests that &ill lower current densities are necessary for optimal resolution.

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covery of the starting material. Figure 4 shows the elution pattern of a fractionation of 100 pmoles of unlabeled dAT oligomers. There was an 85% recovery of the starting material up to fraction 270. Figure 5 shows a photograph of analytical gel patterns of fractions taken from the center peaks 2, 3, and 4 from the preparative experiment depicted in Figure 4. Analyses of fractions taken from various parts of peak 18 are shown in Figure 6. These two figures and other similar experiments suggest that a given fraction contained no more than two species of oligomers at levels perceptible in these analyses. Furthermore, there were fractions in a peak that were essentially free from contamination by material of adjacent peaks. From the elution pattern shown in Figure 4 it can be seen that the relatively sharp leading edge of a peak was likely to be contaminated by material from the more diffuse training edge of the preceding peak. However, once the trailing material was exhausted, essentially uncontaminated oligomer eluted before the appearance of the sharp leading edge of the

FIQ. 5. Analytical electrophoresis of bisacrylamide.

resolution of oligomers Figure 4. The resolving

from peaks 1, 3, and 4 from preparative gel contained 20% acrylamide and 1%

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FIG. 6. Analysis of degree of contamination of fractions from peak 18 of preparative electrophoresis of Figure 4. The resolving gel contained 20% acrylamide and 1% bisacrylamide. Gels were examined after incomplete destaining in order to maximize the detectability of small amounts of contaminants.

succeeding peak. Further evidence for the homogeneity of peak fractions has been provided by sedimentation equilibrium experiments (5). It seems likely that improvements in the technique such as use of lower current density could reduce the trailing of one peak into the nexL6 It should also be noted t,hat t,he resolution of the later peaks was at least as good as that of the earlier ones. The complete specification of the end groups and molecular weighk of the fractionated oligomers is being presented elsewhere (5). The results of these physical and enzymic studies disclose that the degree of polymerization increases by two nucleotides per peak up to values beyond 40 in the later fractions. The method described here has not yet been applied to the fract’ionation of oligomers of mixed base content. However, for the fractionation of homologous oligomere the rrsolring power and capacity of polyacrylamide

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gel electrophoresis seems to justify the effort of the preparative procedure. For example, gel electrophoresis seems to be superior to ionexchange chromatography (20-22) in resolving oligonucleotides of high degree of polymerization. SUMMARY

Polyacrylamide gel electrophoresis has been used to fractionate mixtures of oligomers produced by enzymic digestion of dAT copolymer with pancreatic DNase I. Fractions with degrees of polymerization from about 6 to beyond 40 were obtained with high resolution. ACKNOWLEDGMENTS We are grateful to Dr. L. Stryer for the use of a spectrofluorometer and to Drs. R. L. Baldwin and A. Kornberg for criticism of the manuscript. This work was supported in part by research grants from the U. S. National Institutes of Health (AM 94763) and National Science Foundation (GB 4061). It is based in part on the Ph.D. thesis of Elliot Elson, Stanford University, 1966. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14.

15. 16. 17. 18. 19.

ORNSTEIN, L., Ann. N. Y. Acad. Sci., 121, (2)) 321 (1964). DAVIS, B. J., Ann. N. Y. Acad. Sci., 121, (21, 404 (1964). JOVIN, T., CHAMBACH, A., AND NAUCHMN, M. A., Anal. Biochesm., SCHACHMAN, H. K., ADLER, J., RADDINQ, C. M., LEHMAN, I. R., AND J. Biol. Chem, 235,3242 (1960). SCHEFFLER, I. E., ELSON, E., AND BALDWIN, R. L., J. Mol. BioZ.,‘36, RICHARDSON, C. C., SCHILDKRAUT, C. L., APOSHUN, H. V., AND J. Biol. Chem., 239, 222 (1964). WEBER, G., Advan. Protein Chem., 8, 415 (1953). LERMAN, L. S., Proc. Natl. Acad. Sci. U. S., 49,94 (1963). STRPER, L., J. Mol. Biol., 13, 482 (1965). CLARKE, J. T., Ann. N. Y. Acad. Sci., 121, (2), 321 (1964). WHITE, M., AND DORION, G., J. Polymer Sci., 55, 731 (1961). WHITE, M., J. Phys. Chem., 64, 1563 (19$0). OLIVERA, B. M., BAINE, P., AND DAVIDSON, N., Biopolymers, 2, 245 SMITHIES, O., Advan. Protein Chem., 21, 65 (1959). RICHAWS, E. G., COLL, J. $., AND GIL~TZER, W. B., Anal. Biochem., MCPHIE, P., HOUNSELL, J., .~ND GRATZER, W. B., Biochemistry, 5, GOULD, H., Biochemistry, 5, 1103 (1966). LOENING, 0. E., Biochem. J., 102, 251 (1967). BISHOP, D. H. L., CL.~YBROOE~, J. R., .~ND SPIEGELMAN, S., J. Mol.

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H. G., AND VIZSOLYI, J. P., J. Am. Chem. Sot., 83, 675 (1961). R. V., TENER, G. M., J. A,m. Chem. Sot., 84, 2644 (1962). E., MOON, M. W., AND KHORANA, H. G., J. Am. Chem. Sot., 87, 2956