Volume 5 Number 3 March 1978 Nucleic Acids Research ... · Volume 5 Number 3 March 1978 Nucleic...

Volume 5 Number 3 March 1978 Nuc le ic A c i d s Research

Separation of very large DNA molecules by gel electrophoresis

Walton L. Fangman

Department of Genetics, SK-5Q,University of Washington,Seattle, WA 98195, USA

Received 16 December 1977

ABSTRACT

Very large DNA molecules were separated by electrophoresis in hori-zontal slab gels of dilute agarose. Conditions of electrophoresis weredeveloped using intact DNA molecules from the bacterial viruses X, T4 andG. Their DNAs have molecular weights (M) of 32 million, 120 million, and500 million, respectively. Several electrophoresis conditions were foundwhich give sufficiently high mobilities and large mobility differencesthat these DNAs are separated in a short time. Electrophoresis in 0.1%agarose at 2.5 V/cm of gel length separates TA and A DNAs by 2.0 cm, andG and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilitiesare directly proportional to log M for M values from 10 to 500 million.The procedures,used will allow rapid molecular weight determination andseparation of very large DNA molecules.

INTRODUCTION

The DNA molecules of eukaryotic chromosomes, chloroplasts and large

viruses have molecular weights of 100 million and greater. Such large DNA

molecules cannot easily be separated on the basis of size. Separation by

sedimentation velocity suffers from zone spreading which probably results

from convection, and from the necessity of centrifuging for long periods

at low centrifuge speed. At high centrifuge speeds DNA molecules exceeding

100 million daltons exhibit a marked reduction in s (see citations in

ref. 1). Gel electrophoresis, which has not been applied to very large

DNAs, has been extremely useful for separating smaller DNA molecules (less

than 10 million daltons). Duplex DNA molecules differing by a few percent

in molecular weight can be resolved. A plot of log M versus electrophore-

tic mobility approximates a straight line with large molecules having a

slower rate of migration than smaller ones. However, with the electrophore-

sis conditions usually employed DNA molecules with a mass above 10 million

daltons migrate much faster than expected and are not well resolved.

There have been a large number of publications on the electrophoretic

behavior of small, less than 25 million dalton, DNA molecules (for citations

© Information Retrieval Limited 1 Falconberg Court London W1V5FG England 653

Nucleic Acids Research

see ref. 2). This paper presents conditions for electrophoretic separation

of DNA molecules of 10 to 500 million daltons. The work is an extension of

earlier observations of Henckes et^ a^. (ref. 3). Large DNAs employed in

developing the electrophoresis conditions were the intact molecules from

bacterial viruses X, T4 and G. X and T4 DNAs have been well characterized

and have masses of 32 million and 120 million daltons, respectively.

Bacteriophage G DNA has been reported to have a mass of 500 million daltons

(4). DNA was prepared from bacteriophage G particles by procedures which

eliminate breakage by shear and used as a molecular weight standard.

MATERIALS AND METHODS14 3

Bacteriophage DNA. T4D and XCI857 viruses containing C or H labels

were prepared by standard procedures. Bacteriophage G and its host

Bacillus megatherium PGH were obtained from Prof. G. Donelli. The phage

was cloned and subsequently titred with beef extract broth (BEB) plates

and top agar at 30°. BEB medium contains per liter: 3 g beef extract-5 -4

(BBL), 5 g peptone (Difco) , 5 g NaCl, 8 x 10 moles MgSO,, 2 x 10 moles— 3

MnSO, and 10 moles CaCl., final pH 7.1-7.2. Plates contained 10 g agar/1

and top agar 7.5 g agar/1.- Cells for plating were grown in BEB liquid at

30°. Phage G for DNA isolation was prepared by infecting exponential phase

cells in TYEM medium at a culture optical density (660 nm) of 1.5 withQ

2 x 10 phage per ml of culture. TYEM medium contains per liter: 10 g

tryptone (Difco), 5 g NaCl, 0.5 g yeast extract (Difco) and MgSO,, MnSO,,

and CaCl. as in BEB, final pH 7.4-7.5. With vigorous aeration the culture

lysed in 3-5 hours. The lysed culture was shaken slowly with chloroform

for a few min, then centrifuged at 4,000 x g for 20 min. The supernatant

(1-2 x 10 phage/ml) was centrifuged at 27,000 x g for 30 min and the

pellet containing the phage suspended with cold .01M Tris, .01M MgSO,,

pH 7.4 (TM) containing 1 mg/ml bovine serum albumin (BSA). This material

was incubated with 100 ug/ml RNase I for 15 min at room temperature, then

0.5-1.0 ml (containing 1-5 x 10 phage) was layered onto a cold 16 ml

linear gradient of 10-35% sucrose in TM-BSA. After centrifugation in a

Spinco SW27.1 rotor at 14,000 rpm for 30 min (5°), the phage were collected

as part of a white diffuse band. DNA was labeled by adding 2.5 yc/ml

6- H-uracil at 0, 1 and 2 hours of infection.

DNA was isolated from G particles by very slowly mixing 0.7 ml of

phage suspension with 0.7 ml 10% sodium lauroyl sarcosinate in 0.2M EDTA,

pH 8 in a Spinco SW50 polyallomer centrifuge tube. The tube was corked

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and heated at 65° for 10 min. The solution was underlaid with 3.5 ml room

temperature-saturated CsCl in 0.2M EDTA, pH 8 and centrifuged in an SW50L

rotor at 30,000 rpm for 2-3 days (10°). Fractions were collected through

the tube bottom with a 13G hooded needle (boiled in EDTA solution) at a

flow rate of 0.10-0.15 ml/min. The DNA was dialyzed in a Collodion bag

(S & S) or dialysis tubing held open at one end with a ring of plastic,

against 10 mM Tris, 100 mM EDTA, pH 8 and stored at 5°. For sedimentation

analysis or gel electrophoresis G DNA was kept intact by employing slow

transfers with 1.0-1.5 mm i.d. pipettes. T4 and X DNAs were prepared from

virus particles by the same procedure.

The initial preparations of X and T4 DNAs used in this work contained

intact DNA molecules based on the ratio of their S values and the contour

lengths of the DNAs observed by electronmicroscopy (5, 6). Subsequent

preparations were characterized by electrophoresis in 0.2% agarose as

reported here. The molecular weight of G DNA was determined by cosedi-

mentation with T4 DNA (Figure 3). 3H-labeled G DNA (0.20 ug) and R e -

labeled T4 DNA (0.05 pg) in 0.5 ml 1.0 mM Tris - 1.0 mM EDTA, pH8, was

layered on a 58 ml linear gradient of 15 to 30% sucrose in 0.01M Tris,

pH8 - 0.2M EDTA, pH8 - 0.5M NaCl. The gradient was centrifuged in a

Spinco SW25.2 rotor at 8,000 rpm for 3.5 days (5°). Molecular weights0 38

were calculated from the equation S^S. = (M^M.) " (ref. 7).

Specific fragments of X DNA were generated with the endonucleases Sal

I (New England Biolabs) and Eco RI (supplied by Dr. Maynard Olson and Guy

Page). Sizes for X DNA and X DNA fragments were taken from data of Dr.

Peter Philippsen (personal communication) obtained from electron micro-

scopic contour length measurements. His kilobase pair values were con-

verted to mass units using 660 daltons/base pair to obtain masses, in

millions of daltons, of 32 for intact X DNA, 21 and 10 for the two major

Sal I fragments (A and B, respectively), and 14, 4.9, 3.8, 3.6, 3.1 and

2.2 for the six Eco RI fragments (designated here as A, B, C, D, E, and

F, respectively). T4 DNA was taken to have a mass of 120 million daltons

(8).

Electrophoresis• Horizontal agarose gels were formed on a glass plate

in a plastic container with a raised center section separating two buffer

reservoirs and electrodes. Buffer was in direct contract with the ends of

the gel. Gels were 13.5 cm wide, 10 to 25 cm long and about 1.0 cm thick.

Seakem LE agarose was used for all gels except 0.1% agarose gels in which

Seakem HGT(P) agarose was used to provide greater mechanical stability.

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Gels consisting of less than 0.4% agarose were poured into a "box" of 1%

agarose made by first pouring a 2 nun slab, then four sides 1 cm thick and

1.5 cm wide. This "box" allowed dilute gels to be picked up and moved for

photography and other manipulations. The electrophoresis buffer contained

10.3g Tris, 5.5g boric acid and 0.93g disodium EDTA per liter. For some

gels the buffer contained 4.4 g Tris, 4.1 g anhydrous monosodium phosphate,

0.37 g disodium EDTA per liter. The two buffers gave only small differences

in mobilities. Unless stated otherwise, gels and buffers contained 0.5 yg/ml

ethidium bromide. Sample wells were made with a Biorad teflon comb contain-

ing ten 0.75 x 8.0 mm teeth. The wells were about 8 mm deep and were not

in contact with the 1% agarose bottom layer. Samples of 30 yl were intro-

duced into the wells by slow hand pipetting using a screw-type pipettor and

50 ul disposable glass micropipettes (1 mm i.d.). Gels of 0.1 and 0.2%

agarose were chilled to 5° to stiffen them before removing the comb and

loading the wells. The 30 yl sample was 1 mM Tris, pH 8, 10 mM EDTA, pH 8,

10% glycerol and 0.0015% bromphenol blue and contained 0.05 yg Sal I X DNA

fragments, 0.10 yg Eco RI A DNA fragments, and about 0.025 yg each of X DNA,

T4 DNA and G DNA. Three-fold lower and three-fold higher amounts of the

intact virus DNAs did not result in altered mobilities although streaking

toward the wells occurred at the higher concentrations. The mobility of

each DNA run alone was the same as in the mixture. After loading, the

entire gel was covered with household plastic wrap and electrophoresis was

carried out at room temperature (about 21°) at constant voltage. The vol-

tage gradient was measured with a Midland voltmeter using platinum leads

inserted into the gel at a 10 cm spacing. Gels run without ethidium bro-

mide were subsequently stained by soaking overnight in buffer containing

0.5 yg/ml ethidium bromide. Gels were photographed on a shortwave UV-

illuminator with Kodak Contrast Process Pan Film 4155 through a sandwich of

Wratten No. 9 and No. 25 filters. The exposure time was 4-8 min. Mobilities

were determined by measuring distances on photographs and assuming that the

rate of migration was constant during electrophoresis. The reproducibility

of mobility values was examined in a few cases. These values showed a

standard deviation of ±7% or less. Values reported in the Figures and Tables

are from individual experiments.

RESULTS

Agarose Concentration and Electrophoresis Voltage

The effects of agarose concentration and electrophoresis voltage on

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the separation of DNAs were examined using T4 DNA, X DNA and the restriction

endonuclease-generated fragments of X DNA. Figure 1 and Table 1 show the

effect of varying the agarose concentration (at constant voltage, lV/cm of

gel length) on the absolute and relative mobilities of these DNAs. Although

the absolute DNA mobility (mm/hr) increases as the agarose concentration

decreases from 0.7% to 0.2%, the mobility of each DNA relative to that of

the 14 million molecular weight DNA decreases (Figure 1 ) . This results in

a compaction of the relative mobility distribution for DNAs below this mole-

cular weight. For T4 DNA, X DNA and the 14 million molecular weight DNA,

however, there is a moderate expansion of the relative mobility distribution.

The expansion can be clearly seen as an increase in the X DNA/T4 DNA mobility

ratio at lower agarose concentrations (Table 1 ) . This differential effect,

along with the general increase in absolute mobility, greatly decreases the

IOC

=> I07

o2

10* J I I__L

a b d e —

J I I L _ L _L0-5 1.0 15 2.0 2.5 3.0 3.5

Relative Mobilit y

Figure 1. Effect of agarose concentration on DNA electrophoretic mobility.The voltage gradient was lV/cm of gel length. The lines represent: (a)0.2% agarose without ethidium bromide, (b) 0.2% agarose, (c) 0.3% agarose,(d) 0.4% agarose and (e) 0.5% agarose. Mobilities are normalized to themobility of Eco RI X DNA fragment A which had the following value in eachgel: (a) 5.2 mm/hr, (b) 3.9, (c) 3.3, (d) 1.4 and (e) 1.2. The dashedline is the linear extrapolation of the data for low molecular weight DNAsin experiment a.

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time required to separate X and T4 DNAs (Table 1). Further reduction in

electrophoresis time is obtained by omitting ethidium bromide from the 0.2%

agarose gel; a 1.0 cm separation of T4 and X DNAs requires only 12 hr.

Ethidium bromide was included in most of the gels in this work because this

allows visualization of the DNA during electrophoresis and eliminates the

need to soak the gel in ethidium bromide for 5-10 hr at the end of the

electrophoresis. For preparative work gels can be run without ethidium

bromide and sections of the gel subsequently stained to locate DNA in un-

stained parallel sections.

Figure 2 and Table 2 show the effect of varying the electrophoresis

voltage (V/cm of gel length) on the mobility of these same DNAs in 0.2%

agarose. As the voltage decreases from 2.5V/cm to 0.02V/cm the absolute

mobilities of all the DNAs decrease. There is an expansion of the entire

relative mobility distribution because mobilities of larger DNA molecules

decrease by a larger factor than those of smaller DNA molecules. Most

important, plots of log M versus mobility at lower voltages exhibit a

straight line up to 32 million molecular weight (Figure 2). Separation

of DNAs at lower voltages, of course, is slower. At O.lV/cm a 1.0 cm

separation of T4 and X DNAs would require 92 hr in the presence of ethidium

bromide (Table 2).

Bacteriophage G DNA (500 Million Daltons)

Electrophoretic separation of high molecular weight DNAs was studied

further using DNA from bacteriophage G. This large virus has been reported

Table 1: Effecttion of X and T4

Agaroseconcentration

(%)

0.2a

0.2

0.3

0.4

0.5

0.7

of agarose concentration onDNAs.

Mobility ofT4 DNA(mm/hr)

3.2

2.5

2.2

0.92

0.96

0.53

Data for the 0.2% to 0.5%used to make Figure 1. Voltage

awithout ethidium bromide.

MobilityRatioX/T4

1.25

1.20

1.18

1.19

1.14

1.04

the electrophoretic separa-

Calculatedtime for one cm

separation of X and T4(hr)

12

20

25

57

74

470

agarose gels is taken from the experimentsfor all gels was lV/cm.

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to contain a DNA molecule with a molecular mass and sequence complexity of

approximately 500 million daltons (4, 9). G DNA, isolated as described in

MATERIALS AND METHODS, was analyzed by zone sedimentation. Results such as

those shown in Figure 3 indicate that the procedures employed for isolation

and manipulation of phage G DNA yield a fairly homogenous preparation of

molecules of about 500 x 10 daltons.

Various conditions of electrophoresis were examined with phage G DNA

included as a molecular weight standard. The results of electrophoresis

under conditions which achieve separation of X, T4 and G DNAs in a short

time are summarized in Table 3 and Figure 4. In 0.2% agarose gels run at

lV/cm (experiment d) the T4 DNA/G DNA mobility ratio is 1.10; a 1.0 cm

separation can be achieved in 34 hr of electrophoresis. However, mobility

in the A DNA to G DNA molecular weight range is nonlinear with log M of

the DNA (Figure 4d). Larger DNAs in this range, therefore, would be more

poorly separated under these conditions. Decreasing the agarose concentra-

0.5 1.0 1.5

Relat ive Mob i l i t y

2.0 2 5

Figure 2. Effect of voltage on DNA electrophoretic mobility. The agaroseconcentration was 0.2%. The lines represent: (a) 0.02 V/cm, (b) 0.1 V/cm,(c) 0.5 V/cm, (d) 1.0 V/cm and (e) 2.5 V/cm. Mobilities are normalized tothe mobility of Eco RI A DNA fragment A which had the following value ineach gel: (a) 0.34 mm/hr, (b) 0.64, (c) 2.2, (d) 3.9 and (e) 8.7. Thedashed line is the linear extrapolation of the data for low molecularweight DNAs in experiment a.

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Table 2: Effect of voltage on the electrophoretic separation of X and T4DNAs.

CalculatedMobility of Mobility time for one cm

Voltage T4 DNA Ratio separation of \ and T4(V/cm) (mm/hr) X/T4 (hr)

0.02

0.10

0.50

1.0

2.5

0.11

0.28

1.3

2.5

6.2

1.74

1.39

1.32

1.26

1.15

123

92

24

15

11

The data ia taken from the experiments used to make Figure 2. Allgels were 0.2% agarose.

1200

800 -

EQ.O

400 -

0

- 800

Eexo

- 400

10 20

Froction Number

Figure 3. Sedimentation analysis of bacteriophage G DNA. Details aregiven in MATERIALS AND METHODS. The direction of sedimentation is fromright to left. 100% of the H radioactivity in the G DNA preparation wasin alkali stable material and all of it was recovered in the fractions shownin this figure. Two preparations of G DNA had molecular weights of 510million daltons determined by comparison with. T4 DNA.

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Table 3: Electrophoresis of phage G DNA.

Calculated time forMobility of Mobility one cm separation:

* T4 DNA Ratio X and T4 T4 and GExperiment (mm/hr) X/T4 T4/G (hr)

a 1.1 1.29 1.37 31 34

b 0.90 1.43 1.42 26 38

c (2.3)+ (1.37) (1.36) (<17) (17)

d 3.2 1.27 1.10 12 34

The conditions of electrophoresis were as follows:

Experiment a. 0.1% agarose without ethidium bromide, O.lV/cm, 42 hr.b. 0.1% agarose, O.lV/cm, 41 hr.c. 0.1% agarose, 5V/cm for 1 hr then O.lV/cm for 16 hr.d. 0.2% agarose without ethidium bromide, lV/cm, 21 hr.

Since there was a change in voltage during the electrophoresis themobility is an average value for the total electrophoresis period of17 hr. The actual separations at the end of the electrophoresis were1.5 cm for T4 and X DNAs, and 1.0 cm for G and T4 DNAs.

tion to 0.1% and the voltage to O.lV/cm (experiments a and b) results in an

increase in the T4 DNA/G DNA mobility ratio to about 1.40 and essentially a

straight line for the plot of log M versus mobility for M values from 10 to

500 million (Figure 4a and b). One cm separations of X and T4 DNAs, and T4

and G DNAs can be achieved in 30-40 hr (Table 3). A shift in voltage results

in greater separation in a shorter period. After electrophoresis for 1 hr

at 5V/cm followed by 16 hr at O.lV/cm (experiment c) G and T4 DNAs were

separated by 1.0 cm, and T4 and X DNAs by 1.5 cm. Figure 5 shows densito-

metric tracings of two gels which illustrate the decreased separation of low

M DNAs and increased separation of high M DNAs obtained with 0.1% agarose -

O.lV/cm electrophoresis compared to a 0.2% agarose - lV/cm electrophoresis.

Some skewing of the T4 and G DNA bands toward higher mobility was observed

in 0.1% agarose gels. This may result from degraded DNA molecules or DNA

molecules from petite virus particles found in preparations of T4 (10) and

which may also occur with phage G.

The most rapid separations to date have been achieved using higher vol-

tages with 0.1% agarose, although mobility in the high molecular weight

range is nonlinear with log M of the DNA under these conditions. Figure 6

shows a photograph and tracing of a 0.1% gel (without ethidium bromide)

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10-

10'

10'

0 5

0 5 I O

0.5 1.0I I I

Re la t ive Dis tance

Figure A. Electrophoresis of bacteriophage G DNA. Values for DNAs smallerthan 10 million daltons are not plotted. Gel (a) did not include Sal I XDNA fragments. The electrophoresis conditions are given with Table 3.Distances migrated are normalized to the distance migrated by Eco RI X DNAfragment A in each gel: (a) 7.0 cm, (b) 6.A cm, (c) 6.5 cm and (d) 10.7 cm.

were electrophoresis was at 2.5 V/cm for only 10 hr. TA and X DNAs are

separated by 2.0 cm, and G and TA DNAs are separated by 1.0 cm.

Other Manipulations

Gels as dilute as 0.2% agarose can be handled without great difficulty

when constructed as described in MATERIALS AND METHODS; 0.1% agarose gels

require much more care. Fluoroautoradiograms (11) of H-DNAs have been made

from these dilute gels by partially dehydrating them on a stack of filter

papers, reducing the gel thickness to 2-A mm, before permeating them with

methanol-scintillator solution and drying completely under vacuum. No loss

in resolution was observed. Partially dehydrated gels have also been used

when transfering the DNA to nitrocellulose sheets by the procedure of South-

ern (ref. 12). It should be possible to recover intact duplex DNAs from gel

sections by electroelution.

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D.C

I

iAM,

/

B B'

\

V\

Figure 5. Densitometric scans of two gels. Photographic negatives were Iscanned with a Joyce-Loebl microdensitometer. A' and B' refer to A DNAfragments produced with Sail and A-F to X DNA fragments produced with EcoRI(see MATERIALS AND METHODS).

Left Panel: 0.2% agarose, 1 V/cm, 18 hr (bacteriophage G DNA was 3.5 cmfrom the well; T4/G = 1.12).

Right Panel: 0.1% agarose, 5 V/cm for 1 hr then 0.1 V/cm for 16 hr (bac-teriophage G DNA was 2.9 cm from the well; T4/G = 1.36). This is the samegel as experiment c of Table 3 and Figure h.

DISCUSSION

Linear extrapolations of plots of log M versus electrophoretic mobility

for low molecular weight DNAs at high agarose concentration and high voltage

suggest that mobility would approach zero for larger DNA molecules. This is

not the case; larger molecules migrate faster than expected. Indeed, above

a certain value of M, mobility values for molecules of different molecular

weights are almost the same. Reduction in the agarose concentration and

reduction in the electrophoresis voltage differentially alter the mobilities

of very large DNAs such that approximately linear plots of log M versus

mobility are obtained. The properties of large DNA molecules which result

in these mobility effects may be related to those which result in speed-

dependent sedimentation. This sedimentation effect occurs at low DNA con-

centrations and is observed as a decrease in at higher centrifuge speeds.

Values of s_ approaching that expected from the M value of the DNA are obtained

at low centrifuge speeds. Zimm (ref. 1) has explained this effect as being a

consequence of the extension of large DNA molecules in solution at high speed,

brought about by increased frictional forces at the ends of the molecules.

A similar distortion of DNA by uneven frictional forces during electrophoresis

at higher agarose concentrations and higher voltage gradients (larger force

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Figure 6. Photograph and densitometric scan of a gel. Electrophoresis wasin 0.1% agarose (without ethidium bromide) at 2.5 V/cm for 10 hr. Phage GDNA was 9.4 cm from the well; T4/G = 1.11. The small spike to the right ofG DNA resulted from a piece of lint in the thick gel. See Figure 5 legendfor other details.

on the molecules) generating greater shear stress may result in the rapid

migration of large molecules. x

We can estimate the size difference required to lead to detectable

separation on the gels. The DNA band widths in thex gels are about one mm.

If T4 and G-size DNAs. are electrophoresed so as to be separated by two cm,

molecules in this size range (120 to 500 million daltons) differing in mass

by 30% (mass ratio of 1.3) should be seen as discrete bands. While this is

a great improvement, DNA molecules from 1 to 10 million daltons with mass

differences of only 3% can easily be separated in 0.7% agarose gels run at

lV/cm for 16 hours.

The electrophoresis conditions reported here will be useful for the

analysis and preparation of very large DNA molecules. Molecular weight deter-

minations can be made in a much shorter time than required by sedimentation

because rotor speed distortion of high molecular weight DNA requires long

centrifugations at low speeds. Based on molecular studies (6) and the range

of relative chromosomal lengths indicated by the meiotic karyotype (13), DNA

molecules corresponding to most of the 17 chromosomes of the yeast Saccharo-

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myces cerevisiae are in the molecular weight range spanned by T4 and G DNAs.

Further improvements in the resolution of the electrophoresis should allow

individual chromosomal DNA molecules to be isolated.

ACKNOWLEDGEMENTS

I thank Teri Mallgren for excellent technical assistance and Drs. Breck

Byers and Richard Nelson for their critical reading and thoughtful sugges-

tions for the manuscript. Dr. Donna Montgomery performed the transfer of

DNA from gel to nitrocellulose. This work was supported by grants from the

National Institutes of Health (GM 18926) and the American Cancer Society,

Washington Division (VC-171).

REFERENCES

1. Zimm, B. H. (1974) Biophys. Chem. 1, 279-291.2. Johnson, P. H. and Grossman, L. I. (1977) Biochemistry 16, 4217-4225.3. Henckes, G. , Crochet, M. , Labedan, B. and Legault-Demare, J. (1974)

Anal. Biochem. 60, 1-14.4. Donelli, G. , Dore, E., Frontali, C. and Grandolfo, M. E. (1975) J.

Mol. Biol. 94, 555-565.5. Petes, T. D. and Fangman, W. L. (1972) Proc. Natl. Acad. Sci. USA

69, 1188-1191.6. Petes, T. D., Byers, B. and Fangman, W. L. (1973) Proc. Nat. Acad.

Sci. USA 70, 3072-3076.7. Freifelder, D. (1970) J. Mol. Biol. 54, 567-577.8. Lang, D. (1970) J. Mol. Biol. 54, 567-577.9. Dore, E., Frontali, C. and Grignoli, M. (1977) Virology 79, 442-445.

10. Mosig, G., Carnighan, J. R., Bibring, J. B., Cole, R., Bock, H. 0.and Bock, S. (1972) J. Virol. 9, 857-871.

11. Laskey, R. A. and Mills, A. D. (1975) Eur. J. Biochem. 56, 335-341.12. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517.13. Byers, B. and Goetsch, L. (1975) Proc. Natl. Acad. Sci. USA 72,

5056-5060.

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