COMPUTER-ASSISTED NON-RADIOACTIVE MULTIPLEX GENOTYPING

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The goal of this research is to establish a rapid, non-radioactive multiplex genotyping system for human microsatellite genetic markers. This system will include a computer program for automated allele calling and database interaction. Approximately 300 highly informative microsatellite markers, which are approximately 10 cM apart and which cover their entire human genome, will be identified for multiplex development. Our proposed multiplex system will involve multiplexing at three levels. First, five different markers will be amplified in the same PCR for each individual's DNA. Pooling will be determined based on common PCR conditions. Second, the PCR products from 10 such sets (i.e, the PCR products from 50 loci) will be pooled and separated on denaturing polyacrylamide gels by Direct Transfer Electrophoresis (DTE). The PCR products will be transferred onto nylon membranes for "decoding" by hybridization. Third, the decoding step will be performed using five oligonucleotide probes at each hybridization. These five probes will reveal five individual loci with distinguishable sizes. In addition, the computer program for automated allele calling will feature spatial normalization based on multiplex-specific internal standards, automatic alignment of several images derived form the same membrane, improved algorithms for automatic allele calling, and an intuitive, easily portable user interface. The results of the automatic analysis will be stored in files compatible with commonly used programs for linkage analysis. The finished system will require only six person-months to genotype 300 markers on approximately 200 individuals. The establishment of non-radioactive detection methods is essential for reducing health risks to bench scientists and for eliminating radioactive waste disposal problems.