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, 0305phi8-36. 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 0305phi8-36 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 0305phi8-36 from nonaggregating bacteriophages.

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.

Rapid determination of genomic DNA length for new bacteriophages.

dsDNA viruses with long genomes (>200 kb) are expected to be a major source of novel genes. To rapidly characterize the genomes of newly isolated dsDNA bacteriophages, we develop here a procedure for the PFGE of intact long DNA genomes from bacteriophage particles in unfractionated, infected cell lysates of either liquid or gelled cultures. The DNA used for PFGE is suitable for sequencing after extraction with phenol. The PFGE is tuned to the range of expected DNA lengths. This procedure bypasses the isolation of bacteriophage particles and is useful for PFGE analysis of DNA from dissected zones of bacteriophage plaques.

Matrix conditioning for lengthened capillary DNA sequencing.

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.

Temperature-dependence of preconditioning for lengthened capillary DNA sequencing.

A previous study shows that electrophoretic preconditioning of a commercial polymer solution increases the spacing and resolution of DNA fragments fractionated in this solution by CE at 50 degrees C (Griess, G. A. et al., Electrophoresis 2005, 26, 102). The present study shows that this preconditioning effect on peak spacing progressively increases when the temperature of preconditioning increases to 70 degrees C, though fractionation is still performed at 50 degrees C. An increase in peak sharpness accompanies the increase in peak separation for DNA fragments longer than 200 bases. Changing the preconditioning temperature from 50 to 70 degrees C optimally improves resolution of fragment analysis in the range of 600-2000 nucleotides. When DNA sequencing is performed with automated base calling and 70 degrees C preconditioning at 319 V/cm (47 cm long capillary, Applied Biosystems 310 apparatus), the range of high-quality base calls is increased by 25% to 750; the range of low-quality base calls is increased by about 100% to 1200 in comparison to DNA sequencing without preconditioning.

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.