Electrophoresis of DNA in agarose gels

ANALYTICAL

BIOCHEMISTRY

102, 159- 162 (1980)

Electrophoresis

of DNA in Agarose

II. Effects of Loading Mass and Electroendosmosis PAUL *Department

H. JOHNSON,* of Biochemistry, tDepartment

MICHAEL Wayne

of Cellular The

University

J. MILLER,*

Gels1

on Electrophoretic

AND LAWRENCE

I. GROSSMAN?

School of Medicine, Detroit, Biology, Division of Biological Ann Arbor, Michigan 48109

State University and Molecular of Michigan,

Mobilities

Michigan Sciences,

48201,

and

Received October 2, 1979 We have investigated several experimental factors which affect the accurate determination of electrophoretic mobilities of circular and linear DNAs in agarose gels. We demonstrate that: (1) The mobility of individual DNA species is affected by the total mass in the sample loaded. The increased mobility and band distortion observed become apparent when the DNA mass exceeds approximately 0.2 pg per 0.15 cm* of surface area in the loading well. (2) The migration velocity of a given DNA species depends on the coefficient of electroendosmosis (-m,) of the agarose preparations used. In the range 0.081 s -m, I 0.441, the DNA migration velocity is proportional to (-m ,)-“.S.

Agarose gel electrophoresis allows the rapid separation and high resolution of nucleic acid molecules on the basis of molecular size and conformation (1). Within a limited range, the electrophoretic mobility (CL)of DNA can be an approximately linear function of the logarithm of molecular weight (2-4). In addition, the rate at which the mobility of a molecule changes as a function of gel concentration defines the retardation coefficient, KR (5), a quantity related to the effective mean radius of the migrating molecule (6) Thus, accurate measurment of electrophoretic mobility is required for determination of the various molecular parameters useful for the characterization of DNA structure. In this communication we report that the mobility of individual DNA species is affected by the total mass in the sample loaded and the electroendosmotic properties of the agarose.

MATERIALS

AND METHODS

DNA preparations and the techniques used in performing and evaluating electrophoresis experiments have been previously described in detail (1). Agarose was obtained from Marine Colloids, Inc. (Rockland, Me.) Three preparations of agarose were used which differed in their electroendosmotic properties. Coefficients of electroendosmosis (m,) were supplied by the manufacturer and represent the cathodal movement of a neutral molecule relative to the migration of a standard polyanion. (i) HGT is a high gelling temperature agarose with a low EEO* value (-m, = 0.081), (ii) ME is an agarose of medium EEO (-m, = 0.175), and (iii) HEEO is a high electroendosmosis agarose (-m, = 0.44 1). Densitometric analysis was

’ Paper I in this series is Ref. (1).

159

2 Abbreviations used: HGT, high gelling temperature; EEO, electroendosmosis; ME, medium EEO agarose; HEEO, high electroendosmosis agarose; form I, covalently closed circular duplex DNA; form II, nicked circular duplex DNA: form III, linear duplex DNA.

0003-2697/80/03015904$02.00/0 Copyright D 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

160

JOHNSON,

MILLER,

(-1

(+I

L

AND GROSSMAN

forms are illustrated in Fig. 1, top. Unlabeled form I DNA was added in the indicated quantities to a mixture of 32Plabeled forms I, II, and III (co.05 pg total labeled DNA). As increasing amounts of unlabeled form I DNA were added, all three labeled species migrate more rapidly and as broader and more asymmetric bands. A comparison between the gel tracks containing 0 and 5 pug added DNA is shown as a densitometer tracing in Fig. 1, bottom. Figure 2 shows the distances migrated for the center of the form III band and for the leading and trailing edges of the forms I and II bands versus the amount of unlabeled DNA added to the layered sample. Both the leading and trailing edges migrate further with increasing amounts of added DNA (as do the centers of mass). The effect is greatest for the leading edge, amounting to a difference of 7-8 mm for both forms I and II when the amount of form I DNA added is increased from 0 to 5 pg. The center of mass for the form II DNA band is estimated to have migrated 55% further

FIG. I. The effects of added DNA on electrophoretic mobilities of PM2 DNA conformational isomers. The 5O+l quantities of “*P-labeled PM2 DNA forms (CO.05 pg) containing varying amounts of unlabeled form I DNA (approximately 90% pure) were electrophoresed in a 0.7% agarose slab gel at I .9 V/cm for I5 h. Top: autoradiogram of the dried gel. Bottom: densitometer tracing of gel tracts containing 0 (top) and 5 pg (bottom) of added PM2 form I DNA. The separation distance between forms I and II, in the absence of added unlabeled DNA, is 2.5 cm.

performed using a Model SL-504 Zeineh soft laser scanning densitometer (Biomed Instruments, Inc., Chicago, Ill.). RESULTS AND DISCUSSION

(a) Electrophoretic Mobility of DNA Depends on the Mass in the Sample Loaded The effects of adding increasing amounts of DNA on the mobilities of PM2 DNA

Form I DNA Added

(pq)

FIG. 2. Migration distance of conformational isomers versus total mass in the sample layered. The data from Fig. 3 (top) are represented graphically. Measurements were made from the gel origin to the leading edge of the band (L) and to the trailing edge of the band (T) for forms I and II, and to the band center for form III.

ELECTROPHORESIS

when 5 pg of DNA is added compared to the control sample. This effect can represent a serious source of error when molecular weights are determined from measurements of relative mobility if layered samples of standards and unknowns contain substantially different amounts of DNA. A similar effect was observed by Meyers ef al. (7) for DNA migration in agarose gels, and has been demonstrated during polyacrylamide gel electrophoresis of RNA by Richards and Lecanidou (8). The later study showed that the broadening and increased velocity of the migrating zone is a function of the mass of nucleic acid used, rather than its concentration in the loading well. Our results extend these observations for DNA in agarose gels by demonstrating that these effects take place primarily at the origin, before the conformational isomers have separated, since the pronounced effects on the migrating zone of forms II and III DNA result from the addition of form I (Fig. 1). Figure 2 also indicates that the trailing edge of DNA bands increases less in velocity than does the leading edge as a function of DNA added. Similar results were noted by Richards and Lecanidou (8); this suggests that molecular weight estimates may be more accurately determined by measurements to the trailing edge of DNA bands. However, such measurments involve the error of determining a diffuse boundary. We have, therefore, chosen to measure mobilities from the gel origin to the zone center, using amounts of DNA sufficiently low to avoid these effects (usually less than 0.5 l-4. (b) Effect of Electroendosmosis on the Electrophoretic Migration of DNA Conformational Isomers Electrophoretic mobilities were found to be affected by the electroendosmotic properties of the gel. Figure 3 shows a representation of the migration of +X 174 RF conforma-

OF DNA

161

FIG. 3. Relative separation of DNA conformational isomers in 1% agarose gels of differing electroendosmotic properties. A mixture of 9X174 repicative forms I (-), II (-), and III (-) were electrophoresed for 6 h at 5 V/cm in 1% agarose slab gels (mounted on the same apparatus) having the electroendosmosis values (-m,) shown in Table 1. The diagram represents to scale the ethidium bromidestained I5 cm gel.

tional isomers electrophoresed through each of the three agarose preparations used in this study. Electrophoresis was carried out simultaneously in the divided slab gel apparatus previously described (1). The observed velocities are tabulated in Table 1. In going from an agarose with the highest to the lowest EEO value, the band velocity increased by a factor of 2.1 for form I and III DNA and 2.8 for form II DNA. The larger change for form II DNA alters the relative separation among the three conformational isomers. Thus, form III DNA migrates closer to form II in HEEO agarose and closer to form I in HGT agarose. The increase in band velocity as the -m r value increases is qualitatively expected since the migration of DNA is increasingly being opposed by the cathodal migration of solvent. We observe quantitatively using the data of Table 1 that, in the range 0.087 5 -m, 5 0.441, the migration velocity

162

JOHNSON, TABLE

MILLER,

1

THE DEPENDENCE OF DNA ELECTROPHORETIC MOBILITIES ON THE ELECTROENDOSMOTIC PROPERTIESOF AGAROSE GELS” +X174 RF DNA form

Band velocity (cmimin X 100)

AND GROSSMAN

be used with data for a given gel to predict the migration behavior for an agarose preparation which differs in its electroendosmotic properties. Since migration distance is directly proportional to time (data not shown) the following relationship holds:

Gel

-t?I,h

HGT

0.081

I II III

2.82 1.88 2.28

ME

0.175

I II III

1.92 1.06 1.56

We thank Dr. Greg Petsko, Massachusetts Institute of Technology, for his helpful discussion. This work was supported by NIH Grants GM2369Oand GM21704.

HEEO

0.441

I II III

1.35 0.67 1.09

REFERENCES

” DNA samples were electrophoresed through 1% agarose gels at 5 V/cm. Samples of 4X174 RF DNA were layered every 2 h. Total running time was 8 h. The gel was stained with ethidium bromide and the distance migrated was measured from band center to the bottom of the sample well. Band velocities were obtained from the siope of the computed least-squares line of migration distance vs time. b Values supplied by the manufacturer.

of each +X174 RF species is proportional to (-EQ.)-O.~. Thus, for a given conformational isomer, this linear approximation can

t-2 -= t1

2 (m,,lm,)0~5

[II

2

ACKNOWLEDGMENTS

1. Johnson, P. H., and Grossman, L. I. (1977) Biochemistry 16, 4217-4225. 2. Helling, R. B., Goodman, H. M., and Boyer, H. W. (1974)J. Viral. 14, 1235-1244. 3. Moore, D. H., Johnson, P. H., Chandler, S. E. W., and Grossman, L. I. (1977) Nucleic Acids Res. 4, 1273-1289. 4. Fangman, W. L. (1978) Nucleic Acids Res. 5, 653-665. 5. Ferguson, K. A. (1964) Metubolism 13,985- 1002. 6. Rodbard, D., and Chrambach, A. (1970)Proc. Nat. Acad. Sci. USA 65, 970-977. 7. Meyers, J. A., Sanchez, D., Elwell, L. P., and Falkow, S. (1976)5. Bacterial. 127, 1529-1537. 8. Richards, E. G., and Lecanidou, R. (1971) Anal. Biochem. 40, 43-71.