RAPID SEPARATION OF DNA RESTRICTION DIGESTS USING SIZE-SELECTIVE CAPILLARY ELECTROPHORESIS WITH APPLICATION TO DNA-FINGERPRINTING

Capillary electrophoresis (CE) using running buffers containing a water soluble polymer (methylcellulose, MC) was employed for the rapid size-selective separation and identification of plasmid DNA (pBR322 and pBR328). The two plasmid samples were distinguished based on differences in CE elution patterns resulting from injections of their Hinfl enzyme digests. The restriction fragments in the digests were detected by incorporating the DNA intercalation dye, ethidium bromide (EB), in the running buffer and utilizing laser fluorimetry. Short separation times were achieved by increasing the applied field, but with a concomitant decrease in fragment resolution. This behavior was attributed to three main effects: (i) a reduction in efficiency due to problems with Joule heating, (ii) a decrease in selectivity as the fragments elongate and align with the high fields, and (iii) a general reduction in system retention due to the fragment elongation and increased capillary temperature. The deleterious effects of Joule heating were minimized by employing small diameter capillaries and low buffer concentrations. The molecular weight of the MC polymer primarily influenced separation efficiency. Conversely, MC concentration influenced both selectivity and system retention. High efficiency (10(6) plates m(-1)) and good resolution of the fragments in the different restriction digests were achieved using running buffers containing 100,000 MW methyl cellulose at high fields. Under optimized conditions, the test plasmid sample digest provided reproducible and unique elution profiles with 120 s separation times.