Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

Stellwagen, Nancy C.

doi:10.1002/elps.200500612

Cited by 8 publications

(10 citation statements)

References 39 publications

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“…The Ferguson plots observed for DNA molecules in polyacrylamide gels extrapolate to very different mobilities at zero gel concentration, as shown in Fig. 3 53, 70, 71. If the mobilities observed at zero gel concentration are extrapolated linearly to zero DNA molecular mass, the free solution mobility of DNA is calculated to be (3.1±0.1)×10 −4 cm 2 /V s in 40 mM Tris‐acetate‐EDTA buffer, equal within experimental error to the value obtained from extrapolation of the Ferguson plots observed in agarose gels 86.…”

Section: Dna Interactions With Agarose and Polyacrylamide Gel Matricesmentioning

confidence: 86%

“…However, if preferential interactions of curved DNA molecules with the polyacrylamide gel fibers are responsible for the anomalously slow mobilities, the mobility anomalies should correlate with the acrylamide concentration in the gel, not the apparent gel pore size. When the polyacrylamide gel pore size is decreased by increasing %T at constant %C, the anomalously slow mobilities of the curved DNA molecules increase with decreasing gel pore radius 53, 71, 87, as though sieving effects were responsible for the mobility anomalies 39, 88. However, when the gel pore size is varied by changing %C at constant %T, the anomalously slow mobilities are independent of gel pore radius as long as the apparent gel pore radius is larger than the DNA radius of gyration 87.…”

Section: Dna Interactions With Agarose and Polyacrylamide Gel Matricesmentioning

confidence: 99%

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

2009

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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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Section: Dna Interactions With Agarose and Polyacrylamide Gel Matricesmentioning

confidence: 86%

Section: Dna Interactions With Agarose and Polyacrylamide Gel Matricesmentioning

confidence: 99%

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

2009

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“…Two methods can be used to vary the polyacrylamide gel pore size: changing %T at constant %C, the usual method of changing the pore size, or changing %C at constant %T. When the gel pore size is increased by decreasing %T at constant %C, the mobility anomalies decrease progressively with increasing gel pore radius [139,146,150,160,161]. However, when the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies are independent of gel pore radius as long the pore radius is greater than the radius of gyration of the migrating DNA molecules [146].…”

Section: Polyacrylamide Gelsmentioning

confidence: 99%

“…The combined results indicate that preferential interactions of curved DNA molecules with the polyacrylamide gel matrix, as well as sieving effects, are responsible for the mobility anomalies [146]. The anomalously slow mobilities observed for curved DNA molecules persist even at zero gel concentration [161], because curved DNAs also migrate anomalously slowly in free solution, as will be described below.…”

Section: Polyacrylamide Gelsmentioning

confidence: 99%

Effect of the matrix on DNA electrophoretic mobility

Stellwagen

2009

Journal of Chromatography A

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DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.

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“…Combining electrophoresis and modeling to probe secondary structure in proteins and peptides55, 56 has been mentioned previously. The presence of a gel not only makes it easier to separate DNAs of different lengths, but also DNAs of the same length, but different conformations due to permanent bends or the effect of a bound protein 66–68. These studies are usually carried out in polyacrylamide gels containing crosslinker where the gel structure is nonuniform 69.…”

Section: Discussionmentioning

confidence: 99%

Review modeling the free solution and gel electrophoresis of biopolymers: The bead array‐effective medium model

2007

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Free solution and gel electrophoresis is an extremely useful tool in the separation of biopolymers. The complex nature of biopolymers, coupled with the usefulness of electrophoretic methods, has stimulated the development of theoretical modeling over the last 30 years. In this work, these developments are first reviewed with emphasis on Boundary Element and bead methodologies that enable the investigator to design realistic models of biopolymers. In the present work, the bead methodology is generalized to include the presence of a gel through the Effective Medium model. The biopolymer is represented as a bead array. A peptide, for example, made up of N amino acids is modeled as 2N beads. Duplex DNA is modeled as a discrete wormlike chain consisting of touching beads. The technical details of the method are placed in three Appendices. To illustrate the accuracy and effectiveness of the approach, two applications are considered. Model studies on both the free solution mobility of 73 peptides ranging in size from 2 to 42 amino acids, and the mobility of short duplex DNA in dilute agarose gels are discussed.

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Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

Cited by 8 publications

References 39 publications

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

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

Effect of the matrix on DNA electrophoretic mobility

Review modeling the free solution and gel electrophoresis of biopolymers: The bead array‐effective medium model

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