SENSITIVITY, REPRODUCIBILITY, AND ACCURACY IN SHORT TANDEM REPEAT GENOTYPING USING CAPILLARY ARRAY ELECTROPHORESIS

The Human Genome Initiative has increased significantly the rate at wh ich disease-causing genes are being mapped and sequenced. New cost-eff ective methods to locate the genes and to characterize disease-causing mutations require robust, reproducible, and accurate protocols For me asuring DNA fragment lengths. Capillary array electrophoresis (CAE) of fers rapid, high-resolution separations, high throughput, and sensitiv e detection. To assess the utility of CAE For the accumulation of gene tic information, we tested both sizing accuracy and reproducibility us ing 48-capillary prototype systems. Two multiplex PCR allelic ladder s tandards and several CA-repeat markers were analyzed in >100 runs. Rep roducibility 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, resultin g 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 1 bp of actual. We detect as little as a 1:30,000 dilution of a DNA quantitat ion standard stained with highly sensitive intercalating dyes, indicat ing an 80-zeptomole sensitivity limit. However, to obtain reproducible electrokinetic injection, similar to 200 attomoles of fluorescein-lab eled DNA is required. These sensitivity limits, sizing precision, and accuracy, together with the 1-hr run times For 48-96 samples, indicate that CAE is a viable method for high-throughput genetic analysis of s imple sequence repeat polymorphisms.