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Review
. 2009 Jun;30 Suppl 1(Suppl 1):S188-95.
doi: 10.1002/elps.200900052.

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

Nancy C Stellwagen  1
Affiliations
Review

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

Nancy C Stellwagen. Electrophoresis. 2009 Jun.

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 primarily due 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) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees 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.

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Conflict of interest statement

The author has declared no conflict of interest.

Figures

Figure 1
Figure 1
Electrophoresis of normal and anomalous DNA fragments in: (A), 2.0% agarose gels; and (B), 5.7%T, 1.5%C polyacrylamide gels. Monomers of normal (N) and anomalous (A) DNA restriction fragments containing 167 bp were ligated (separately) to create multimers of various sizes. From bottom to top, successive bands in each lane of each gel correspond to monomers, dimers, trimers, 4-mers, 5-mers and higher multimers of the normal (N) and anomalous (A) DNAs. The effective pore radius of the agarose gel is estimated to be 51 nm [69]; the effective pore radius of the polyacrylamide gel is estimated to be 132 nm [83].
Figure 2
Figure 2
Ferguson plots observed for normal DNA molecules in agarose gels. The logarithm of the mobility, extrapolated to zero electric field strength at each gel concentration, is plotted as a function of agarose concentration, %A. The lines were drawn by linear regression; the size of each DNA, in kilobase pairs, is given beside each line. Adapted from [51] with permission.
Figure 3
Figure 3
Ferguson plots observed for normal (Δ) and anomalous (○) DNA fragments in polyacrylamide gels. The logarithm of the mobility is plotted as a function of polyacrylamide concentration, %T, in gels containing 3%C. The normal and anomalous fragments contained one, two, four or eight monomers, from top to bottom; the size of each pair of fragments, in kilobase pairs, is indicated beside each pair of lines. All lines were drawn by linear regression. Adapted from [101] with permission.
Figure 4
Figure 4
Free solution mobility of normal (open circle) DNA fragments of various sizes in 40 mM Tris-acetate-EDTA buffer, plotted as a function of the number of base pairs in each fragment. The closed circles indicate the mobilities of curved 199-bp fragments containing 1 – 5 A-tracts in a curvature module located in the center of each fragment. Adapted from [110] with permission.
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