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 fluorescenc e (CE-LIF) detection. phi X-174 DNA was digested with HaeIII restricti on enzyme under conditions that allowed the monitoring of digestion as it proceeded toward completion. Separation by a polymer solution of m ethylcellulose in a polyacrylamide coated capillary allowed high resol ution 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 u sing ethidium bromide as an intercalation dye and allowing intercalati on to occur on-column. It is demonstrated that the course of the diges tion (i.e., the creation and diminishing of fragment peaks) can be fol lowed 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 contai ns II potential restriction endonuclease cleavage sites. This initial attempt to follow a restriction digest on-column not only provides mea ningful information for the biochemical researcher, but also furthers the use of CE as a diagnostic tool for the biochemical laboratory. (C) 1997 Elsevier Science B.V.