Controlled fragmentation of DNA by DNase I

ANALYTICAL

Controlled ANNA HELL, The

48, 364-377 (1972)

BIOCHEMISTRY

Beatson

Fragmentation cr. D. BIRNIE,

Institute

for

Cancel

of DNA ‘I’. Ii. SI,I?\I?\IING,

Research,

Rweiwd

1%

Hill

Street,

by DNase 4ND

Glasgow,

I

JC)HK PArI, C’S, Scotland

Deccmbcr 1, 1Dil

In preliminary experiments designed to isolate unique sequencesfrom mammalian DNA (1)) we found it was not possible to obtain a clean fractionation of single-stranded and double-st,randed material on hydroxyapatite columns (1) with DNA of average single-stranded molecular weight 250,000 or great’er. The importance of DNA size for the rate of DNA reannealing has been clearly demonstrated by Wetmur and Davidson (2). Moreover, it may be necessary with eukaryotic DNA to use fragments small enough to permit the separation of repeated and nonrepeated sequences which may be short and adjacent to each other. It appeared, therefore, that for studies of the hybridization of DNA and RNA from eukaryotic cells, DNA fragments of approximately the same size as a gene (say 400 to 500 nucleotides in length) are desirable. We were unable to obtain DNA fragments of molecular weight lower than 250,000 by physical met’hods. This paper describes the conditions for controlled shearing of DNA by DNase I and subsequent isolation of segments of known molecular weight in large quantities. MATERIALS

AND

METHODS

Preparation of DNA The method is based on that of Britten et al. (3). Nuclei were isolated from 15-18 day old mouse embryos by homogenizing the whole embryos in 10 vol 0.025 M citric acid at 4”. Nuclei were pelleted by centrifuging at 1OOOgfor 10 min. The pelleted material was washed by rehomogenizing it in 4 or 5 portions of 0.025 111citric acid (10 vol each) and once in 0.0025 M citric acid, recentrifuging each time at 1OOOgfor 10 min. The nuclear pellet was suspended in 20 vol 8 M urea/O.24 M sodium phosphate buffer, pH 6.8 (MUP), containing 1% (w/v) SDS and 0.01 M EDTA. It was blended in a sealed, filled cont’ainer with an MSE homogenizer run at 14,000 rpm for 6 periods of 15 set each, alternating with 30 set cooling in ice. The homogenate was poured into a thick 369

@ 1972 by Aratlcmic

Press, Inc.

370

HELL

ET

AL.

slurry of hydroxyapatite (HAP) (Bio-Rad Bio-Gel HTP) in MUP, and the mixture was stirred gently and allowed to stand at room temperature for about 1 hr. (The amount of HAP used varied according to the capacity of the particular batch of HAP available.) The slurry was poured into Pyrex No. 3 sintered-glass funnels (approximately 30 gm HAP per funnel of diameter 9.5 cm) and the liquid was drawn off under a vacuum of 5 psi. The HAP was washed free of RNA and protein with MUP until no further material was eluted as judged by E,,, and E,,, measurements on the effluent (approximately 1500 ml/30 gm HAP), then with a further 500 ml MUP. Urea was removed by washing with 0.014M sodium phosphate, pH 6.8 (approximately 500 ml required/30 gm HAP). The removal of urea was monitored by refractive index measurements on the effluent. The DNA was eluted with 0.4M sodium phosphate, pH 6.8. The DNA solution was dialyzed for 18 hr against 10 vol distilled water at 4”; then solid NaCl was added to 0.2 M. DNA was precipitated with 2 vol ethanol at -20” for 18 hr. The precipitate was collected by cenkifugation at 16,3008 for 30 min, dried in air at room temperature, and redissolved in distilled water (the precipitate contains considerable salt). This DNA4 solution was dialyzed three ti,mes against 100 vol 0.05 M NaC1. DNA so prepared was heavily contaminated with Ca?+ (up to 2 mg Ca”+/lOO mg DNA). It was decalcified by stirring for 15 min with Dowex 50 Na+ (Bio-Rad analytical-grade cation-exchange resin AG 5OW-X8, 1 gm resin/50 mg DNA). The slurry was poured into a column containing 1 cm of packed Dower; 50 Na+ and the resin was washed with 3-5 ml 0.05 M NaCl. Ca’+-free DNA was recovered quantitatively in the total effluent. DNA obtained in this way was of relatively uniform size, varying little from one preparation t’o another. The native molecular weight was 2.3 to 3.5 X lOG and the denatured molecular weight was 0.9 to 1.4 X 106. It contained less than 0.5% RNA (alkali-labile ,material) and less than 1% protein (4). The ratios E260/EZ30= 2.4, and E2&EZZ0 = 1.8 to 1.9. This method has also been used to prepare DNA from mouse liver, Landschutz ascites-tumor cells, and LS cells. In each case 1 gm wet weight nuclei yielded approximately 10 mg DNA. DNase DNase I, RNase-free, was obtained from Worthington Biochemical Corp. It was dissolved at 20 pg/ml in 1 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, from Sigma Chemical Corp.), adjusted to pH 7.0 with KOH, and filtered through a sterile Millipore

FRAGllIENTATION

371

OF DN.4

filter (0.45 p). It was mixed with an equal volume of sterile glycerol, sealed in 1.0 ml portions in sterile ampules, and stored at -20”. This solution retained at least 7570 of its original activit’y over 6 months. For each experiment a fresh ampule was taken and the whole contents appropriately diluted witll 1 mJ1 HEPES, pH 7.0. F,raymentation

of DNA

DNA (400 PgJml) was dissolved in 0.1 M NnQ’0.02 M MgSO,/O.l M HEPES, pH 7.0. DNase was diluted to twice the required final concentration in I mM HEPES, pH 7.0. The solutions were warmed at 37”) mixed in equal volumes, and incubated for 2 hr at 37”. All pipets and tubes used were of sterile disposable plastic or acid-washed glass. The reaction in incubation mixtures up to 1.4 ml in volume was stopped by heating for 15 min at 60”. The solution was desalted by centrifuging it through a column of 10 ml Sephadex G-25 (fine) (5). Reactions on a preparative scale were stopped by cooling in ice and stirring with Dowex 50 Na+ (about 1 gm/20 ml incubation mixture) for 15 min. The slurry was poured into a column containing 1 ml packed Dowex 50 Na’. The effluent. was collected and the remaining DNA eluted with 0.05 M NaCl. To denature the DNase, the solution was made 0.2iM with respect to NaOH and, after 15 min, neutralized with HCl at 4” with vigorous stirring. DNA was precipitated with 2 vol ethanol at -20” for 18 hr. Deterneination

of

Molecular

Weight

of DNA

Molecular weights were determined from the sedimentation rates of DNA in sucrose gradients using the conditions of Studier (6). To find the molecular weight of DNA prior to degradation, linear 5-20s (w/w) sucrose gradients were used ; neutral gradients for native DNA contained 1 M NaCl/O.Ol X Tris-HCX, pH 8; alkaline gradients for denatured DNA contained 0.9 M NaCI/O.l M NaOH. To determine the molecular weight of small DNA fragments, 5-10s (w/w) sucrose gradients were used. Such shallow gradients are necessary to obtain the resolution required in this low molecular weight region (7). Samples were sedimented through either 14 ml gradients in the MSE 6 X 15 swingout rotor, or through 20 ml gradients in the MSE 3 X 23 swingout rotor, in MSE superspeed 65 or 50 ultracentrifuges. Fractions (0.75 or 1.0 ml, respectively) were collected by upward clisplacemcnt by 30% (w/w) sucrose in 1 M NaCl. The sedimentation coefficients were determined using tables of McEwen (8) and, from them, molecular weights were calculated using equations given by Studier (6) :

372

HELL

ET

AL.

for native DNA in neutral gradients, s’?~,~ = 0.0882 M”.X6 for denatured DNA in alkaline gradients, s”~~,~ = 0.0528 J!L!o.400 Ltrrge-Scale

Fractionation

in n Zonnl Rotor

A titanium BSIV zonal rotor was loaded with a 500 ml 5-20s (w/w) sucrose gradient linear with rotor radius. The gradient was prepared in apparatus similar to that described by Birnie and Harvey (9). The mixing vessel contained 600 ml 5% (w/w) sucrose to which was added 500 ml 30% (w/w) sucrose. The sucrose solutions contained 0.9ill NaCI/O.l M NaOH. The sample (10 ml) of fragmented DNA in 0.9 M NaCl/O.l M NaOH was overlaid with 100 ml 0.9 M NaCl/O.l M NaOH. The rotor was spun at 45,000 rpm for 17 hr at 20”. RESULTS

Determination

of Molecular

Weight of DNA

F,raqme?ds

It became evident that the relationship between sedimentation coefficient in alkaline sucrose gradients and molecular weight of DNA fragments as defined by Studier (6) depended on the tot,al absence of Ca’+ and POb3- (Table 1). Both Ca?+ and Pod”- caused marked increases in sedimentation coefficient. When Mg?+ is present in the samples, the precipit,ate of Mg(OH)* formed in the alkaline gradient absorbs much of the DNA, which is t’hen pelleted. In view of these results Ca”+, Mg’+, and POb3- were rigorously excluded from DNA samples before they were sedimented through alkaline sucrose gradients.

Effect

Ion ___-_ Ca*+ p(&::-

of Cat+

and POa3-

TABLE on Sedimentation

Concn. in sample

s”qw

1 of DNA

in Alkaline

of DNA

Sucrose

Gradients

Apparent stranded

singlemol. wt.

~
M

0.06 M 0,125 iI1 0.25 Ar

Frugmedution

12.5

0.8

x 106

14 3

1.2

x x

10”

10 x

104

2.5 104 2..5 X 104 4 x 104

3 4 5.3

of DNA

by IjNase

The rate at vvhic,h DNA is degraded by low concentrations of DNase decreases rapidly within 1 hr and thereafter the reaction proceeds slowly (Fig. 1). The standard incubation time of 2 hr was chosen, therefore, to minimize variations in the sizes of the DNA fragments produced.

FRAGMENTATION

L OO

I 1

I time

373

OF DNA

I 2

I 3

(hours)

FIG. 1. Time course of degradation of DNA by DYase I. DNA with DNase at 5 mpg/ml and samples vverc taken at intervals for of mol. wt. 1 ml samples were analyzed on 14 ml alkaline 510% gradients, spun in a 6 X 15 swingout rotor in an MSE superspeed 65 at 24,500 rpm for 41 hr at 20”.

was incubated determinat’ion (w/w) sucrose ultracentrifuge

0. 3

0. 2

t

8 2 0‘.l

15 IO

oj 5 01

FIG. 2a. DNA fragmentation over a range of DNase concentrations. The distribution of mol. wts. obtained at DNase concentrations of 10 mpg/ml (A--A) ; 5 mpg/ml (0-O); 3 mpg/ml (a---@); 1 mpg/ml (A-A) (top panel); bottom panel shows the sucrose gradient (O-O), .~“2~.~ value (0 ... o), and mol. wt. (A- -A) plots. One ml samples were analyzed on 14 ml alkaline 510% (w/w) sucrose gradients in a 6 X 15 ml swing-out rotor in an MSE superspeed 65 ultracentrifuge at 24,500 rpm for 36.5 h at 20”.

374

HELL

DNase

ET AL.

concentration

(mpg/ml)

FIG. 2b. DNA fragmentation over a range of DNase concentrations. Peak mol. wts. obtained in four separate experiments, over a period of one month, using portions of the same DNase solution stored in 1 mM HEPES/50% glycerol at -20”. The different symbols represent the different experiments. All incubations were for the standard time of 2 hr.

The relationship between DNase concentration and molecular weight of DNA fragments produced is shown in Figs. 2a-b. A narrower dist.ribution of molecular meight is obtained in the more highly degraded samples (Fig. 2a). The results from a series of experiments such as that illustrated in Fig. 2a are summarized in Fig. 2b, which shows the degree of reproducibility obtained. Preparation and Isolation of Milligram

Amounts of DNA Fragments

Results of experiments on an analytical scale indicated that it was possible to fragment DNA to a predicted small size with DNase. However, as shown by Fig. 1, the size of DNA fragments so produced covers a rather wide range. A preliminary experi,ment showed it was possible to fragment large amounts (about 20 mg) of DNA with DNase and isolate fragments of narrowly defined size range by fractionation of the sheared DNA on an alkaline sucrose gradient in a zonal rotor. This was confirmed and extended by the experiment illustrated by Figs. 3a-b. Three portions of DNA, each of 15 mg, were treated with different concentrations of DNase calculated (Fig. 2b) to produce DNA fragments of molecular weight ranging from 70,000 to 300,000. After incubations were stopped the reaction mixtures were pooled, and the mixture of DNA fragments was sedimentcd through a 5-2070 (w/w) alkaline sucrose gradient in a tit,anium BXIV zonal rotor. Figure 3a shows that the DNA fragment,s so produced covered a very wide range of molecular weights. The material in the gradients was fract,ionated as indicated in Fig. 3a and portions of these fractions were sedimented through analytical gradients. The distribution of DNA fragments in each fraction is

FRAGMENTATION

OF

DNA

375

FIG. 3a. Isolation of milligram amounts of DNA fragments in alkaline 520% (w/w) sucrose gradient in titanium BXIV zonal rotor: (0) distribution of DNA in the rotor, (0) the sucrose gradient. Rotor was spun at 45,000 rpm for 17 hr at 20”.

25

A BC Y ** .

DEF *++

FIG. 3b. Isolation of milligram amounts of DNA fragments in alkaline 520% (w/w) sucrose gradient in titanium BXIV zonal rotor. The analytical gradients on samples from the zonal fractions marked by arrows in Fig. 3a. Top panel: (A) O--O; 03) A--n; (0 n . . em; (D) O--O; (E) A-A; (F) 0.. .O. Bottom panel shows sucrose gradient (-), ~“~0,~ value (0 .** O), and mol. wt. (A--& plots. 1 ml samples were analyzed on 14 ml alkaline 5-10s (w/w) sucrose gradients, spun in a 6 X 15 ml swingout rotor in an MSE superspeed 65 ultracentrifuge at 24,700 rpm for 39.5 hr at 20”.

376

HELL

ET AL.

shown in Fig. 3b. The fragment sizes in these fractions cover relatively narrow ranges, especially compared to those found in individual reactions such as shown in Fig. 2a (Table 2). TABLE Fragment

Sample 1.

2.

A B C A B C D E F

Sizes

2

Obtained from Individual and from Zonal Fractions

DNased (2)

Samples

(1)

Peak mol. wt. (x 10-3)

R/Iol. wt. range at half peak height (X 1OV)

so 200 350 90

30-300 50-500 SO-600 36-160 75-300 175-400 260-500 340-660 430-725

150

250 350 450 500

DISCUSSION

Many methods for preparing DNA from eukaryotic cells have been described. That of Britten et al. (3) was chosen for this work because it gives a very reproducible product, in terms of yield, molecular weight, and purity. The yield is almost quantitative and so the possibility that specific fractions of DNA are lost during the preparation is much reduced. The uniformity in molecular weight is important for the reproducibility of fragmentation by given concentrations of DNase. In addition, the met.hod allows preparat’ion of quantities of the order of 50 to 100 mg very rapidly, and with minimal contamination wit’h RNA and protein. It is known that the action of DNase I on DNA is to cause singlestrand breaks (10). Preliminary experiments confirmed this mechanism under our conditions, about 20 single-strand breaks occurring for each double-strand break. This meant that only estimations of the molecular weight of single-strand molecules were meaningful. However, this is no disadvant,age, since physical methods of fragmenting DNA to small molecular weight segments, such as high-pressure shearing, also cause variable degrees of denaturation (1). Moreover, denatured DNA is required for hybridization experiments. In our hands, physical methods of shearing DNA did not yield segments of average single-stranded molecular weight much less than 250,000. FragmentaGon of DNA by DNase I readily allows DNA to

FRAGMEKTATION

OF DNA

377

be sheared to single-stranded segments in a wide range of molecular weights in a controlled manner. Since the sedimentation rate of DNA from any organism is relatecl to the molecular weight of the DNA rather than to its buoyant density (11)) mixtures of DNA fragments can be fractionated according to size on sucrose density gradients. Thus, sedimentation of DNase-sheared DNA through an alkaline sucrose gradient in a zonal rotor enables denatured DNA fragments of narrowly defined size ranges to be isolated in useful amounts. SUMMARY

Large amounts of purified DNA have been prepared by a method based on that of Britten et al. (3). This DNA is of relatively uniform molecular weight and can be sheared reproducibly by DNase I to yield small molecular weight fragments. Large-scale preparations using alkaline sucrose gradients in a titanium BXIV tonal rotor yield milligram amounts of DNA fragments of defined size range. A\CKNOWLEDGMEKT This work was supported by grants from the Medical Cancer Research Campaign.

Research Council and the

REFERESCES 1. BRITTEX, Ii. J., AND KOHNE, D. E., Science 161, 529 (1968). 2. WETMUR, J. G.. AKD DAVIDSON, N., J. MoZ. Biol. 31, 349 (1968). 3. WRITTEN, R. J., PAVICH, &I., AND SMITH, J., Carnegie Inst. Wushington Yea&. 68, 400 (1969). 4. LOWRY, 0. H., ROSEBROUGII, K. J., FARR, h. L., AND RAKD.+LL, R. J., J. I&or. Chem.

193,

265

(1951).

5. DETERMANN, H., in “Gel Chromatography,” p, 58. Springer, Berlin, 1968. 6. STUDIER, F. W., J. Mol. Biol. 11, 373 (1965). 7. BIRNIE, G. D., HELL, A., SLIMMING, T. Ii., AND PAUL, J., in “Methodological Developments with Zonal Rotors” (E. Reid, ed.). Longmans, in press. 8. McEwsx, C. R., Awl. Rio&em. 20, 114 (1967). 9. BIRNIE, G. D., AND HARVEY. D. R., Awzl. Biochem. 22, 171 (1968). 10. YOUNG, E. T.. II, AND SINSHEIMER, R. I,.. J. Biol. Chem. 240, 1274 (1965). 11. HALSAI,L, H. B., AKD SCHVM.ABER, V. N., iC’ature 221, 772 (1969).