Low molecular weight carboxylic acids are separated and quantitated by capillary zone electrophoresis (CZE) with an on-column conductivity detector. The addition of 0.2-0.5 mM TTAB (tetradecyltrimethylammonlum bromide) controls the electroosmotlc flow so that all carboxylate anions pass through the detector. Unlike other CZE detection methods, conductivity detection shows a direct relationship between retention time and peak area. This confers on conductivity detection, In CZE, the unique advantage that use of an Internal standard allows accurate determination of absolute concentrations In a mixture without separate calibration of response for each component.

Fluorescently labeled DNA fragments generated in enzymatic sequencing reactions are rapidly separated by capillary gel electrophoresis and detected at attomole levels within the gel-filled capillary. The application of this technology to automated DNA sequence analysis may permit the development of a second generation automated sequencer capable of efficient and cost-effective sequence analysis on the genomic scale.

A major challenge of the Human Genome Initiative is the development of a rapid, accurate, and efficient DNA sequencing technology. A major limitation of current technology is the relatively long time required to perform the gel electrophoretic separations of DNA fragments produced in the sequencing reactions. We demonstrate here that it is possible to increase the speed of sequence analysis by over an order of magnitude by performing the electrophoresis and detection in ultra thin capillary gels. An instrument which utilizes these high speed separations to simultaneously analyze many samples will constitute a second generation automated DNA sequencer suitable for large-scale sequence analysis.

DNA sequence analysis is based upon the electrophoretic separation of DNA fragments by denaturing polyacrylamide gel electrophoresis. It is necessary to understand the factors which determine resolution in these separations in order to optimize performance. Resolution is determined by the width of the DNA bands and the spacing between the bands. In this paper, resolution is studied using capillary gel electrophoresis in a nonstacking buffer system. It is shown that the width of the DNA bands is determined by four factors: injection, diffusion, thermal gradients, and detection volume. The relative contribution of each effect is determined as a function of fragment length. This information is used in conjunction with empirically determined mobilities to predict resolution as a function of capillary length at a fixed electric field. The implications of this analysis for optimum separations are discussed.

Capillary gel electrophoresis (CGE) has demonstrated the ability to separate DNA sequencing reactions at speeds up to 25 times as great as conventional slab gel electrophoresis. These increased speeds are made possible by the efficient heat dissipation of capillaries, which permits higher electric fields to be employed without deleterious thermal effects. The high electric fields, however, also lead to a reduction in the spacing between bands with a concomitant loss of resolution. The resulting tradeoff between speed and resolution is a very important practical aspect of these high-field separations. This work addresses this question by investigating the band broadening and resolution of DNA fragments as they are separated through a fixed distance of gel at field strengths ranging from 50 to 400 V/cm. It is found that the bandwidths of DNA fragments do decrease with the higher field strengths due to a reduction in the diffusional broadening of bands. However, at sufficiently high electric field strengths, the bands begin to broaden again due to the thermal gradient across the gel. This behavior causes the optimum electric field strength for maximum fragment resolution to depend upon the length of fragments being separated. The relative contributions of diffusion and thermal gradients are discussed and used to predict the ultimate performance of constant field capillary gel electrophoresis.