We have developed a procedure for synthesizing large stable branched DNA structures that enables visualization via fluorescence microscopy. Using this procedure we have synthesized large DNA stars and observed their electrophoretic behavior in polymer solutions and gels. In dilute polyacrylamide solutions, the DNA stars move as random coils and appear to experience only brief collisions with the polymer chains in solution. The effect of polymer solution concentration on the electrophoretic mobility of stars in the dilute regime is found to be in good accord with predictions of the transient entanglement coupling (TEC) model. In semidilute polymer solutions, the star arms extend in the field direction and drag the core through the matrix. The star arms form several U-shaped conformations as they collide and engage with polyacrylamide chains. The U-shaped conformations occasionally evolve into J-shaped conformations as the star arms slide off the matrix chains they engage during electrophoretic migration. In concentrated polymer solutions, the arms of the star extend and form V-shaped structures with the core as the apex. The arms then pull the core through the matrix. These V-shaped conformations are much longer-lived than U-shaped ones and, unlike the latter, do not transform to J-shaped conformations. In polyacrylamide and agarose gels, where matrix entanglements are fixed, DNA stars become trapped when entanglements with matrix molecules prevent the core from being pulled through the matrix by the extended arms. This trapping was observed at all gel concentrations and electric fields studied.

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.

The electrophoretic mobility of three-arm asymmetric star DNA molecules, produced by incorporating a short DNA branch at the midpoint of rigid-rod linear DNA fragments, is investigated in polyacrylamide gels. We determine how long the added branch must be to separate asymmetric star DNA from linear DNA with the same total molecular weight. This work focuses on two different geometric progressions of small DNA molecules. First, branches of increasing length were introduced at the center of a linear DNA fragment of constant length. At a given gel concentration, we find that relatively small branch lengths are enough to cause a detectable reduction in electrophoretic mobility. The second geometric progression starts with a small branch on a linear DNA fragment. As the length of this branch is increased, the DNA backbone length is decreased such that the total molar mass of the molecule remains constant. The branch length was then increased until the asymmetric branched molecule becomes a symmetric three-arm star polymer, allowing the effect of molecular topology on mobility to be studied independent of size effects. DNA molecules with very short branches have a mobility smaller than linear DNA of identical molar mass. The reason for this change in mobility when branching is introduced is not known, however, we explore two possible explanations in this article. (i) The branched DNA could have a greater interaction with the gel than linear DNA, causing it to move slower; (ii) the linear DNA could have modes of motion or access to pores that are unavailable to the branched DNA.

Electrophoresis of large linear T2 (162 kbp) and 3-arm star-branched (N(Arm) = 48.5 kbp) DNA in linear polyacrylamide (LPA) solutions above the overlap concentration c* has been investigated using a fluorescence visualization technique that allows both the conformation and mobility mu of the DNA to be determined. LPA solutions of moderate polydispersity index (PI approximately 1.7-2.1) and variable polymer molecular weight Mw (0.59-2.05 MDa) are used as the sieving media. In unentangled semidilute solutions (c* < c < c(e)), we find that the conformational dynamics of linear and star-branched DNA in electric fields are strikingly different; the former migrating in predominantly U- or I-shaped conformations, depending on electric field strength E, and the latter migrating in a squid-like profile with the star-arms outstretched in the direction opposite to E and dragging the branch point through the sieving medium. Despite these visual differences, mu for linear and star-branched DNA of comparable size are found to be nearly identical in semidilute, unentangled LPA solutions. For LPA concentrations above the entanglement threshold (c > c(e)), the conformation of migrating linear and star-shaped DNA manifest only subtle changes from their unentangled solution features, but mu for the stars decreases strongly with increasing LPA concentration and molecular weight, while mu for linear DNA becomes nearly independent of c and Mw. These findings are discussed in the context of current theories for electrophoresis of large polyelectrolytes.

The electrophoretic mobility of three-arm star DNA structures with varying degrees of branch length asymmetry has been investigated in polyacrylamide (PAA) hydrogels. We report the effect of single-base mismatches, adjacent to the branch point, on the mobility of branched DNA with three different arm lengths. Branched DNA structures were formed using wild-type and mutated fragments of the p53 tumor suppressor gene, which is believed to play an important role in cancer development. Branching was directed at the site of several previously characterized mutations in exon 7 of p53. At a given gel concentration, the mobility of branched DNA with fully complementary base pairing is found to increase as the degree of branch length asymmetry is increased. Ferguson analysis of the gel electrophoresis data leads to a retardation coefficient that is strongly dependent on topology. This finding can be explained in terms of a minimum molecular cross-section for each molecule. Specifically, we show that structures with the smallest molecular cross-section can access more pores in the gel, which leads to higher mobility. Our results can also be understood by considering the rotational diffusivity of branched DNA. Asymmetric DNA stars with higher calculated rotational diffusivities also have higher mobilities. When a mutated base is present in junctions with low degrees of branch length asymmetry, adjacent to the branch point, the mobility increases in comparison to the fully complementary molecules. The reason for this increased mobility is unclear, here, we propose that the mismatched base introduces additional flexibility to the arm containing the mutation leading to higher conformational freedom and enhanced mobility in gels. When a mismatched base is present in junctions with high degrees of branch length asymmetry, the opposite result is obtained. Here, the mutated species has a lower mobility. This result is argued to arise from incomplete hybridization and/or frayed ends. Finally, we have shown that by using two of the branch point oligonucleotides as probe molecules, mutations known to occur at specific sites can be detected through the mobility shift. If the sequences of the probe chains are changed in a controlled manner, the location and base of the mutant can also be determined.