The high-resolution separation of double-stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high-resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp-40 kbp) of DNA fragments without the use of pulsed fields. We used a low-viscosity sieving medium (ca. 5 cP, at 25 degrees C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Mn 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Mn 8 x 10(6)) to separate fragments between 1-40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high-throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.

The Human Genome Initiative has increased significantly the rate at which disease-causing genes are being mapped and sequenced. New cost-effective methods to locate the genes and to characterize disease-causing mutations require robust, reproducible, and accurate protocols for measuring DNA fragment lengths. Capillary array electrophoresis (CAE) offers rapid, high-resolution separations, high throughput, and sensitive detection. To assess the utility of CAE for the accumulation of genetic information, we tested both sizing accuracy and reproducibility using 48-capillary prototype systems. Two multiplex PCR allelic ladder standards and several CA-repeat markers were analyzed in >100 runs. Reproducibility in typing >8000 genotypes reveals a standard deviation of less than 0.2 bp on these systems under optimized conditions. However, sequence-dependent migration anomalies were observed at most simple sequence loci even when analyzed under denaturing conditions, resulting in a systematic bias in estimated fragment sizes. We show here that, by normalizing results to known typing controls, one can obtain locus-averaged accuracies of <0.06 bp and normalized results within I bp of actual. We detect as little as a 1:30,000 dilution of a DNA quantitation standard stained with highly sensitive intercalating dyes, indicating an 80-zeptomole sensitivity limit. However, to obtain reproducible electrokinetic injection, -200 attomoles of fluorescein-labeled DNA is required. These sensitivity limits, sizing precision, and accuracy, together with the I-hr run times for 48-96 samples, indicate that CAE is a viable method for high-throughput genetic analysis of simple sequence repeat polymorphisms.

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