The Electrophoretic Mobility of DNA in Agarose Gel as a Function of Temperature

ROGER WEST, Medical Research Coucil, Mammalian Genome Unit, Kings Buildings, West Mains Road, Edinburgh, Scotland

INTRODUCTION

Variations of temperature within gels are a well-known nuisance in gel electrophore- sis, leading to distortion of banding patterns. The variation of the gel electrophoretic mobility of polyions as a function of temperature is therefore a matter of practical interest.

EXPERIMENTAL METHOD AND RESULTS The apparatus comprised eight separately water-jacketed, vertical, thin-walled glass

tubes mounted between an upper common cathodic tank and a lower common anodic tank. The water jackets were maintained at temperatures between 15 and 50° C by rapidly circulating water from water baths. The tubes had an internal diameter of 6 mrn and were 298 mm long, of which 230 mm was within the water jacket. Rods of 1.5% w/v agarose were confined well within the water-jacketed portions of each tube and the temperature of the gel after equilibration was assumed to be that of the water as it left the water jackets. The electrophoresis buffer, with which the gels were in equilibrium, was 30 millimolar sodium phosphate, 1 millimolar EDTA, pH 6.8 at 2 2 O C. A large volume of this buffer was continually circulated between the two electrode tanks and a reservoir. Each gel was loaded with 0.3 g of bacteriophage X DNA completely digested with restriction endonuclease Ava I, and electrophoresis was for 16 h at 1.07 v/cm. After electrophoresis the gels were stained with ethidium bromide and photographed under uv light through a red filter: the distance moved by each band was measured from the photograph. The mobilities of the fragments are plotted vs temperature in Fig. 1. Above about 45OC the mobility declined sharply, and the 50° C point has been omitted from Fig. 1. I believe this decline occurs because partial melting of the DNA and/or the agarose increases the hydrodynamic interaction between the DNA and the gel.

If Fig. 1 is compared with a plot of the reciprocal viscosity of water vs temperature,' it is evident that a large part of the temperature coefficient of electrophoretic mobility may be ascribed to the variation in the viscosity of the electrophoresis buffer over the experimental range of temperature. The remaining discrepancy is probably due to the temperature coefficient of the chain flexibility of DNA molecules, and of the ionic dissociation of polyelectrolytes. The physical principles of both these factors are well understood? Any plausible theory of gel electrophoresis would have to treat these variables according to known principles, and it is therefore unlikely that temperature effects will be of much value in discriminating between alternative theories of gel electrophoresis. I hope, however that the information presented here may enable others to make empirical corrections for temperature variation in agarose gel electro- phoresis of DNA. I t is clear from the figure that, within the temperature range of 15-45OC, the ratio of the mobilities of a DNA molecule at two temperatures, TIoC and T'O C, is approximately:

mT1 TI + 30 mT2 T2 + 30 _ _ = ~

Biopolymers, Vol. 26, 607-608 (1987) 0 1987 John Wiley & Sons, Inc. CCC oooS-3525/87/050607-02$04.00

608 BIOPOLYMERS VOL. 26 (1987)

TEMPERATURE OC

Fig. 1. Electrophoretic mobility of DNA in agarose gel plotted vs temperature ( O C). For experimental details, see text. The lines through the points are calculated by the least-squares method and extrapolated to - 30 O C. The molecular sizes of the DNA species are (top to bottom): 1000,1040,1160,2250,2890,3590,4354,4480,5930,6490, and 7990 base pairs.

References

1. Weast, R. C., Ed. (1984) Handbook of Chemistry and Physics 65th ed., Chemical Rubber

2. Bloomfield, V. A., Crothers, D. M. & Tinoco, I. (1974) Physical Chemkby of Nucleic Acids, Publishing Company, h a Raton, FL.

Harper & Row, New York.

Received June 6,1986 Accepted November 24,1986