RAPID SIZING OF POLYMORPHIC MICROSATELLITE MARKERS BY CAPILLARY ARRAYELECTROPHORESIS

Genetic mapping and DNA sequencing projects could potentially be compl eted more rapidly by using capillary array electrophoresis (CAE) syste ms running 48-96 capillaries simultaneously, Currently, multiplex poly merase chain reaction (PCR) and multicolor fluorescent dye labeling st rategies are used to generate DNA profiles containing 18-24 genotypes per sample. By using 4-color fluorescence detection and these multiple x PCR strategies, a CAE system has the capacity to generate up to 5.5 million genotypes per year. CAE offers extremely fast, high-resolution separation of DNA and more automated sample processing than conventio nal systems because the labor-intensive slab-gel pouring and sample-lo ading steps are eliminated. We used a prototype CAE system in an ongoi ng linkage analysis study of inherited deafness in Mediterranean famil ies, CA-repeat markers linked to deafness susceptibility genes on chro mosomes 7, 11 and 13 were analyzed and DNA profiles generated which co ntain 6 markers per color. Fragment sizes of over 28000 short tandem r epeat alleles and 3200 CA-repeat alleles have been determined by CAE. An average sizing precision of +/- 0.12 base pairs (bp) for fragments up to 350 bp was realized in 1-h runs. In addition, a versatile non-de naturing matrix was used to separate DNA sizing standards, restriction digests, and multiplex PCR samples. Application of this matrix to Duc henne muscular dystrophy exon deletion screening is also described. Th ese CAE approaches should facilitate rapid genotyping of microsatellit e markers and subsequent identification of disease-causing mutations. (C) 1997 Elsevier Science B.V.