Sensitivity, reproducibility, and accuracy in short tandem repeat genotyping using capillary array electrophoresis.
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 1 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, approximately 200 attomoles of fluorescein-labeled 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 simple sequence repeat polymorphisms.
Mansfield
E S
ES
Molecular Dynamics, Inc., Sunnyvale, California 94086, USA. emansfield@mdyn.com
Vainer
M
M
Enad
S
S
Barker
D L
DL
Harris
D
D
Rappaport
E
E
Fortina
P
P
D002197:Capillary Action; D004247:DNA / Q000032:analysis*; D018900:Dinucleotide Repeats; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D005819:Genetic Markers; D005838:Genotype; D016045:Human Genome Project; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000235:genetics*; D012015:Reference Standards; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D004247:DNA