Dispersion of solute by electrokinetic flow through post arrays and wavy-walled channels.

In this work, we simulate electrokinetically driven transport of unretained solute bands in a variety of two-dimensional spatially periodic geometries (post arrays as well as sinuous/varicose channels), in the thin Debye layer limit. Potential flow fields are calculated using either an inverse method or a Schwarz-Christoffel transform, and transport is modeled using a Monte Carlo method in the transformed plane. In this way, spurious "numerical diffusion" transverse to streamlines is completely eliminated, and streamwise numerical diffusion is reduced to arbitrary precision. Late-time longitudinal dispersion coefficients are calculated for Peclet numbers from 0.1 to 3162. In most geometries, a Taylor-Aris-like scaling law for the dispersion coefficient D(L)/D(L0) = 1 + Pe2/alpha underpredicts dispersion when Pe approximately O(alpha1/2) (here D(L0) is the effective axial diffusion coefficient in the periodic geometry). A two-parameter correlation widely used in the porous media literature, D(L)/D(L0) = 1 + Pe(n)/alpha, agrees slightly better, but much better agreement can be obtained using a new quadratic form: D(L)/D(L0) = 1 + Pe/alpha1 + Pe2/alpha2. A quasi-universal relationship for stream-wise dispersion is offered that predicts 96% of the simulation data to within a factor of 2 in all geometries studied. Comparison with previous work shows that in circular post arrays, the dispersion coefficient for electrokinetic flow is a factor of 3-10 less (depending on Pe and relative post size) than for pressure-driven flow.

In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs.

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-micrometer microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry approximately 10(6) strands complementary to one of the tags. About 10(5) copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Motions of short DNA duplexes: an analysis of DNA dynamics using an EPR-active probe.

The dynamics of a series of four DNA duplexes of length 12, 24, 48, and 96 base pairs have been studied using an electron paramagnetic resonance (EPR) active nitroxide spin-label covalently attached to a thymidine located near the center of each duplex. The linear EPR spectra were simulated by solving the stochastic Liouville equation for anisotropic rotational diffusion. The diffusion tensor for global rotation of the duplex was predicted from hydrodynamic theory for a right circular cylinder. All internal motions, assumed to be rapid, are modeled by reduced electron Zeeman and hyperfine tensor anisotropies. Best fit simulations to the data were then obtained by adjusting the total amplitude of all internal dynamics. The local, length-independent and the collective, length-dependent contributions to the internal dynamics were separated by determining the total amplitude of internal motion as a function of duplex length. The major axis of the spin tensors was determined to be tilted 20 degrees from the helix axis. As a result, the spin-label is most sensitive to flexural motions of the DNA duplex. It is found that the global tumbling of duplex is accurately modeled by hydrodynamic theory. The length-independent motion is characterized by a root-mean-squared amplitude of oscillation of 10 degrees in two dimensions at 20 degrees C and has a strong temperature dependence, indicating that the local structure of the DNA changes with temperature. The length dependence of the internal dynamics leads to an estimate of the dynamic flexural persistence length of 2500 +/- 340 A. There was no statistically significant difference between models assuming a harmonic or a square-well local bending potential.

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.

Octanol-water partition coefficients of substituted alpha, N-diphenylnitrones and benzonitrile N-oxides.

Experimental octanol-water partition coefficients are reported for substituted alpha, N-diphenylnitrones and benzonitrile N-oxides. The results of these measurements are used to calculate the aromatic fragment constants pi HC = N(O)C6H5, pi C = N----O, fHC = N(O), and fC = N----O for the group contribution methods of Hansch and Leo.