The greater negative charge density of DNA in tris-borate buffers does not enhance DNA condensation by multivalent cations.

As indicated by recent measurements of the electrophoretic free solution mobility, DNA appears to have a greater helical charge density in Tris-borate-EDTA (TBE) buffers than in Tris-acetate-EDTA (TAE) buffers. Since electrostatic forces play a major role in DNA packaging processes, we have investigated the condensation of closed circular plasmid DNA using total intensity and dynamic light scattering in Tris-borate, Tris-acetate, and Tris-cacodylate buffers with cobaltic hexa-amine (III) [Co(NH(3))(3+)(6)]. We find that neither the critical concentration of Co(NH(3))(3+)(6) nor the hydrodynamic radii of the resulting condensates vary significantly in the buffer systems studied here despite the prediction that DNA condensation should occur at significantly lower Co(NH(3))(3+)(6) concentrations in Tris-borate buffers. Assuming a persistence length behavior similar to B-DNA in the presence of multivalent cations, a decrease in the attractive counterion correlation pressure decay length in Tris-borate buffers does not account for our observations. It is possible that the binding of multivalent cations to DNA may hinder borate association with the DNA double helix.

Electrophoretic separation of linear and supercoiled DNA in uncoated capillaries.

We report electrophoretic separation of supercoiled plasmids (2-16 kilo base pairs) and linear double-stranded DNA (0.6-23 kilo base pairs) in uncoated capillaries filled with dilute hydroxyethylcellulose. Because electroosmotic flow reverses the order of elution, long plasmids spend less time in the capillary and their bandwidths are narrower than observed in coated capillaries. However, resolution is similar to that obtained in coated capillaries, because it is governed by the distribution of unresolved topoisomers. In the presence of electroosmotic flow migration of supercoiled plasmids does not follow the elastic rod model that has been observed in coated capillaries.

Band broadening during dc and field inversion capillary electrophoresis of lambda DNA in dilute hydroxyethylcellulose solutions.

Capillary electrophoresis of lambda double-stranded (ds) DNA (48.5 kbp) in dilute hydroxyethylcellulose (HEC) solutions shows band spreading that cannot be explained by diffusion alone. Dispersion and asymmetry factors of lambda ds-DNA bands were measured as functions of capillary length, HEC concentration and field strength. Band spreading and asymmetry can be explained by a recently developed model in which the dominant contribution is assumed to be dispersion in DNA-HEC disentanglement times. Bandwidth reduction using square-wave field inversion was also investigated. It is proposed that correlation of DNA motion is the source of band narrowing during field inversion.

High-speed separation of linear and supercoiled DNA by capillary electrophoresis. Buffer, entangling polymer, and electric field effects.

Capillary electrophoresis in dilute and semidilute (slightly entangled) hydroxyethyl cellulose (HEC) is shown to separate linear double-stranded DNA (ds-DNA) and supercoiled plasmid DNA in the size range 1-16 thousand base pairs in 3 min. The mobilities of linear ds-DNA fragments are stronger functions of electric field strength and buffer concentration than the mobilities of supercoiled plasmids. The effects of HEC concentration and molecular weight are similar for both forms of DNA. The behavioral differences, which are attributed to the greater stiffness of the plasmids, can be used to define conditions that maximize resolution of supercoiled and linear ds-DNA of the same or similar number of base pairs.

Capillary electrophoresis of supercoiled and linear DNA in dilute hydroxyethyl cellulose solution.

Capillary electrophoresis in dilute hydroxyethyl cellulose is shown to separate supercoiled DNA in the size range 2000-16,000 base pairs. The plasmids migrate more slowly than linear ds-DNA of the same sizes. Plasmid bandwidths are larger than observed for ds-DNA, allowing identification of the type of DNA by bandwidth. The differing dependence of mobility on chain length can be explained by assuming that a plasmid migrates as an elastic rod, while ds-DNA migrates as a wormlike chain.

Fluorescence resonance energy transfer visualization of DNA aggregates formed during electrophoretic separations in ultradilute hydroxyethylcellulose.

Fluorescence resonance energy transfer (FRET) microscopy is used to observe structures of DNA aggregates formed during electrophoresis in ultradilute hydroxyethylcellulose (HEC). FRET gives evidence of aggregates with a tightly packed core and random entanglement of DNA in the outer portions. HEC concentrations also appear to play a role in aggregate formation. DNA aggregates take longer to assemble in higher HEC concentration solutions and assume a less compact form than those formed at lower HEC concentrations.