ULTRAFAST DNA ANALYSIS BY CAPILLARY-ELECTROPHORESIS LASER-INDUCED FLUORESCENCE DETECTION

The limits of ultrafast DNA analysis by CE were determined by investig ating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 b p). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to min imize the extra column effects on peak dispersion. Two major column di spersion factors, logitudinal diffusion and thermal dispersion, were d etermined experimentally, as well as the influence of the electric fie ld strength on the electrophoretic mobilities and diffusion coefficien ts of DNA. It was found that higher field strengths can be applied wit h lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction d ue to field-induced DNA orientation. Short capillaries (similar to 3-7 cm effective length) and moderate to high electric field strengths (s imilar to 600-800 V/cm) were used to perform a series of fast DNA sepa rations. The dsDNA fragment standards Phi X174/HaeIII and pBR322/HaeII I were separated within 30 s. The possibility for fast mutation detect ion was demonstrated using constant denaturant capillary electrophores is (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.