The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from Ϸ6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D ϳ L ؊ was observed with L ‫؍‬ 0.571 ؎ 0.014, C ‫؍‬ 0.589 ؎ 0.018, and S ‫؍‬ 0.571 ؎ 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of Х 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio DCircular͞ DLinear ‫؍‬ 1.32 ؎ 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model. circular ͉ polymer ͉ polyelectrolyte ͉ hydrodynamics ͉ excluded volume W hile many eukaryotic genomes are linear, prokaryotic genomes and most cloned DNA constructs are circular (1). Indeed, a commonly stated motivation for theoretical calculations on circular polymers is that they may be applicable to understanding the behavior of DNA. However, four of the five previously reported studies on the diffusion of circular polymers have used synthetic polymers, and only two of these, both using synthetic polymers, examined the dependence of the diffusion coefficient on molecular length. The dependence of D on length for relaxed circular DNA has never been measured. Here, we examine linear, relaxed circular, and supercoiled DNA molecules covering a wide range of lengths (Ϸ6 to 290 kbp).For long linear polymers in a good solvent, where excluded volume effects are appreciable, polymer physics theory (2) predicts D ϳ 1͞R G ϳ L Ϫ with Х 0.588, where R G is radius of gyration. The same scaling exponent has been calculated for both dynamic (D ϳ L Ϫ ) and static (R G ϳ L ) quantities, with nearly identical results determined by using a wide range of methods. Static scaling has been examined by using Monte Carlo simulations (3), bead-rod simulations (4), and cylindrical selfavoiding polygon models (5). Renormalization group theory methods have been used in both static (3, 6) and dynamic (7) calculations, and bond-fluctuation simulations (8) were used to measure and compare both scaling relationships. The predicted scaling exponent is close ...

Imaging of single DNA molecules has enabled detailed studies of dilute polymer dynamics and rigorous testing of assumptions and predictions of molecular theories. It is of interest to extend these methods to the study of entangled polymers and to correlate molecular dynamics with rheology measurements. Progress in this direction has been hampered, however, by a lack of available DNA samples in sufficient quantities and covering a wide range of lengths. Here we describe the preparation of a suitable set of molecules ranging in length from ∼3 to 300 kilobase pairs. These constructs are replicated as plasmids or as fosmids or bacterial artificial chromosomes fitted with an inducible high-copy number origin of replication. DNA sequences were chosen to allow molecules to be linearized by single-cutting restriction enzymes. We show that these molecules can be imaged and characterized by fluorescence microscopy and can be prepared in sufficient quantities for bulk rheology measurements.

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Recent trends in the development of microfluidic and biodiagnostic chips favor polymer materials over glass, primarily for optical and economical reasons. Therefore, existing chemical methods to prepare biomolecule microarrays on glass slides have to be adapted or replaced in order to suit polymer substrates. Here we present a strategy to immobilize DNA and antibodies on cyclic polyolefin slides, like Zeonor. This polymer represents a class of new polymeric materials with excellent optical and mechanical properties. By plasma and liquid chemical treatment followed by coating with polyelectrolytes, we have succeeded in immobilizing DNA onto the polymer substrate, yielding stable and versatile biosensor surfaces. We demonstrate the stability and usage of the coated Zeonor substrates not only in DNA chip technology but also in protein chip technology with DNA-directed immobilization of proteins.

Real-time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface-selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow-fragment-catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.