Gary A. Griess

The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases below 1.5%, the semilogarithmic F versus Pc plot becomes progressively less linear. Two straight lines represent the data; the plot is steeper at the lower Pc values. As the percentage of agarose increases above 1.5%, the semilogarithmic F versus Pc plot becomes steeper at the higher Pc values. This change in the shape of semilogarithmic F versus Pc plots is possibly explained by the existence in agarose gels of two zones, one whose Pc distribution is more sensitive to the average agarose concentration than the other. To compare the structure of agarose gels to their sieving during electrophoresis, the root mean square value of Pc (Pc) is compared to the sieving-based radius of the effective pore (PE; Griess et. al. (16)) for both underivatized agarose and a derivatized agarose that has a smaller PE at any given agarose percentage. For 0.8-2.0% gels of either underivatized or derivatized agarose, PE/Pc is a constant within experimental error. Deviations from this constant are observed at lower gel percentages. This relationship of PE to Pc constrains theoretical descriptions of the motion of spheres in fibrous networks.

Non-denaturing gel electrophoresis of biological nanoparticles : viruses

Although gel electrophoresis is usually used for the fractionation of monomolecular particles, it is also applicable to the fractionation of the multimolecular complexes produced during both cellular metabolism and assembly of viruses in virus-infected cells. Gel electrophoretic procedures have been developed for determining both the size of a spherical particle and some aspects of the shape of a non-spherical particle. Capsids bound to DNA outside of the capsid can also be both fractionated and characterized. The procedures developed will be used for screening viral mutants; they also can potentially be used for diagnostic virology. Sensitivity of detection, the major current limitation, is being improved by use of both improved stains and scanning fluorimetry. The gels used for fractionation sometimes approximate random straight fiber gels, but become increasingly biphasic as the gel concentration is decreased.

Determination of a particle's radius by two-dimensional agarose gel electrophoresis.

Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles. Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility. Accuracy of 0.3 nm has been achieved with particles 30 nm in radius. It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid. Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles.

Advances in the separation of bacteriophages and related particles

Nondenaturing gel electrophoresis is used to both characterize multimolecular particles and determine the assembly pathways of these particles. Characterization of bacteriophage-related particles has yielded strategies for characterizing multimolecular particles in general. Previous studies have revealed means for using nondenaturing gel electrophoresis to determine both the effective radius and the average electrical surface charge density of any particle. The response of electrophoretic mobility to increasing the magnitude of the electrical field is used to detect rod-shaped particles. To increase the capacity of nondenaturing gel electrophoresis to characterize comparatively large particles, some current research is directed towards either determining the structure of gels used for electrophoresis or inducing steric trapping of particles in dead-end regions within the fibrous network that forms a gel. A trapping-dependent technique of pulsed-field gel electrophoresis is presented with which a DNA-protein complex can be made to electrophoretically migrate in a direction opposite to the direction of migration of protein-free DNA.

Unlimited increase in the resolution of DNA ladders.

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two‐step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double‐stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back‐and‐forth migration. Improved resolution of DNA bands is achieved, without a known limit.

Application of the concept of an electrophoretic ratchet

Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R ≥ 95 nm) spheres (Serwer, P., Griess, G.A., Anal. Chim. Acta 1998, 372, 299—306). The electrical oscillations are the following electrical field pulses: high field → low field → high field, etc. The field is inverted after each pulse; the time‐integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field‐direction, but not in the low field‐direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field‐direction, when the above‐described pulsed field is biased in the high field‐direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field‐direction. Here, the R of ratchet‐responding spheres is reduced to 30—50 nm. These ratchet‐responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet‐responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet‐based procedure is developed here to achieve continuous preparative gel electrophoresis.

In situ fluorescence microscopy of bacteriophage aggregates

Virus aggregation is analyzed because of its potential use for both classifying viruses and understanding virus ecology and evolution. Virus aggregation is, however, problematic because aggregation causes loss of virions during processing for microscopy of any type. Thus, here we detect virus aggregation by fluorescence microscopy of wet‐mounted dissections of dilute gel‐supported plaques (in situ fluorescence microscopy) of a test virus, the long‐tail aggregating Bacillus thuringiensis bacteriophage, 0305φ8–36. Background fluorescence is reduced to nonproblematic levels by using the dye, DAPI (4′,6‐diamidino‐2‐phenylindole), to stain viral nucleic acid. In situ fluorescence microscopy reveals two in situ phases, one weakly fluorescent. Most bacteriophages partition to the weakly fluorescent phase. Aggregates of bacteriophage 0305φ8–36 are detected during fluorescence microscopy via the following. (1) Coordinated motion is found for visibly separate particles in solution; the motion is either thermally generated, fluid drift‐induced or mechanical pressure‐induced. (2) Aggregate dissociation is observed. Some of the larger aggregates are elastic and entangled with material of the weakly fluorescent phase. The larger aggregates sometimes sink at 1‐g within specimens. In situ fluorescence microscopy rapidly distinguishes 0305φ8–36 from nonaggregating bacteriophages.

Cyclic capillary electrophoresis.

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re‐analysis that is done in several stages of constant‐field electrophoresis. Enhancement‐stages are between the analysis‐stages. An enhancement‐stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement‐stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero‐integrated. The ratchet‐generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion‐induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement‐stages are critically dependent on the electrical field‐dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer‐controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet‐generating pulses. These other pulses can be either the previously used, shorter field‐inversion pulses or high‐frequency periodic oscillations previously found to sharpen peaks.

Routine fluorescence microscopy of single untethered protein molecules confined to a planar zone

To bypass limitations of ensemble averaging biochemical analysis, microscopy‐based detection and tracking are needed for single protein molecules that are diffusing in aqueous solution. Confining the molecules to a planar zone dramatically assists tracking. Procedures of microscopy should be routine enough so that effort is focused on the biochemistry. Fluorescence microscopy and partial planar confinement of single, untethered, aqueous protein molecules have been achieved here by use of a routine procedure. With this procedure, multiple thermally diffusing Alexa 488‐stained bovine serum albumin molecules were observed during partial confinement to a thin aqueous zone next to a cover slip. The procedure produces confinement by partial re‐swelling of a previously dried agarose gel on the microscope slide. Confinement was confirmed through analysis that revealed thermal motion lower in the third dimension than it was in the plane of observation.

Improving the length-fractionation of DNA during capillary electrophoresis.

The present study develops a path‐lengthening strategy for capillary electrophoresis of short double‐stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two‐stage cycles (cyclic electrophoresis): The first analysis‐stage of each cycle is a constant field (forward) capillary electrophoresis. This analysis‐stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement‐stage of each cycle is zero‐integrated field electrophoresis (ZIFE). The enhancement‐stage improves the DNA length‐fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement‐stage. Increase in both peak separation and peak sharpness contribute to improvement in the length‐fractionation of DNA molecules.