SHORT TANDEM REPEAT TYPING BY CAPILLARY ARRAY ELECTROPHORESIS - COMPARISON OF SIZING ACCURACY AND PRECISION USING DIFFERENT BUFFER SYSTEMS

Polymorphic microsatellite markers are widely used in gene discovery a nd mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these m arkers requires polymerase chain reaction (PCR) amplification and very -high-resolution electrophoresis. Capillary array electrophoresis offe rs extremely fast, high-resolution separation of DNA and more automate d sample processing because labor-intensive slab-gel pouring and sampl e loading are eliminated. We report a simple, reliable procedure for p reparing PCR samples for electrokinetic injection into capillaries usi ng a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the siz ing accuracy and precision of low-viscosity, replaceable matrix formul ations. Our study sizing over 28,000 alleles yielded an average precis ion of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulati ons permit low-pressure matrix injection (40 psi) and a turnaround tim e of 70 min for 48-96 samples. (C) 1997 Academic Press.