Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

Abstract

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are due primarily to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 ± 0.01) × 10 -4 cm 2 /Vs in 40 mM Tris-acetate-EDTA buffer at 20°C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.Keywords agarose gels; capillary electrophoresis; DNA; free solution mobility; polyacrylamide gels Historical overviewThe study of DNA electrophoresis began in 1964, when three groups of investigators [1][2][3][4][5] measured the mobility in free solution using moving boundary methods. They found that the mobility was independent of size for DNA molecules larger than ∼400 base pairs (bp) [5], and varied with ionic strength [3,5] and the identity and valence of the cation in the background electrolyte [2,3]. At about the same time, inspired by the separation of proteins in synthetic gel matrices [6][7][8][9][10], other investigators began to use similar matrices to separate RNA [11][12][13][14][15][16][17][18][19] and DNA molecules [15,[20][21][22][23][24] by molecular mass. The separation matrices included agar [11,18,20,21], agarose [22,23], polyacrylamide [13,14,19,[24][25][26][27][28] and composite agarose-acrylamide [16,17] gels. As electrophoretic methods were improved by the purification of agarose [29][30][31] and the use of slab gels instead of tube gels [25,32], and as the discovery of restriction enzymes allowed the preparation of monodisperse DNA fragments of known size [33,34], it became apparent that the separation of DNA fragments by molecular mass depended on the gel matrix in which the separation was carried out Correspondence: Dr. Nancy C. Stellwagen, Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA, nancystellwagen@uiowa.edu, Fax: +319-335-9570. The author has declared no conflict of interest. NIH Public Access Author ManuscriptElectrophoresis. Author manuscript; available in PMC 2010 June 1. [33,35]. Electro...

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