SIMULTANEOUS ASSESSMENT OF LOSS OF HETEROZYGOSITY AT MULTIPLE MICROSATELLITE LOCI USING SEMIAUTOMATED FLUORESCENCE-BASED DETECTION - SUBREGIONAL MAPPING OF CHROMOSOME-4 IN CERVICAL-CARCINOMA

Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considera ble advantages over traditional Southern blotting-based approaches. Ho wever, current methodologies are limited by several factors, including the numbers of loci that can he evaluated for LOH in a single experim ent, the discrimination of true alleles versus ''stutter bands,'' and the use of radionucleotides in detecting PCR products. Here we describ e methods for high throughput simultaneous assessment of LOH at multip le loci in human tumors; these methods rely on the detection of amplif ied microsatellite loci by fluorescence-based DNA sequencing technolog y, Data generated by this approach are processed by several computer s oftware programs that enable the automated linear quantitation and cal culation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 w ere determined for 58 carcinomas of the uterine cervix. The results un derscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.