Temporal analysis of DNA restriction digests by capillary electrophoresis.

We demonstrate a facile means for temporal analysis of DNA restriction enzyme digests by capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection. phi X-174 DNA was digested with HaeIII restriction enzyme under conditions that allowed the monitoring of digestion as it proceeded toward completion. Separation by a polymer solution of methylcellulose in a polyacrylamide coated capillary allowed high resolution and a high degree of reproducibility between sequential runs. At pre-selected time intervals an injection of the digest, directly from the reaction mixture, was made. Sensitive detection was achieved by using ethidium bromide as an intercalation dye and allowing intercalation to occur on-column. It is demonstrated that the course of the digestion (i.e., the creation and diminishing of fragment peaks) can be followed using this methodology. Also demonstrated is the ability to use temporal analysis to determine ideal conditions for producing a single cut within a cloning and expression vector (pET3a-PAI-1) which contains 11 potential restriction endonuclease cleavage sites. This initial attempt to follow a restriction digest on-column not only provides meaningful information for the biochemical researcher, but also furthers the use of CE a diagnostic tool for the biochemical laboratory.

Design and optimization of a capillary electrophoretic mobility shift assay involving trp repressor-DNA complexes.

An investigation of DNA-protein interactions by capillary electrophoresis (CE) with laser fluorometric detection is performed that combines the rapid and minimal sample consumption methods of CE with the selective separation influence of mobility shift assays. An inspection of the well characterized interaction between the trp repressor of Escherichia coli and the trp operator (DNA) is the basis of the assay. The use of fluorescently tagged operator not only lends itself to laser-induced fluorescence detection but also precludes the use of radiolabeled detection. It is demonstrated that composition and pH of the running buffer are critical for maximized efficiency and resolution of operator from the repressor-operator complex. Quantitative studies showed reaction of repressor with operator resulted in the diminishing of free operator signal and the simultaneous creation of the repressor-operator peak that is well resolved from the free operator. Also examined was the ability to perform qualitative studies involving non-specific interactions between the operator and a complex protein sample. It is shown that the specificity of operator for repressor can be used to selectively separate the repressor from a complex sample that includes non-specific proteins.