We will explore the feasibility of a genetic mapping system using polymorphic sequence-tagged sites (pSTSs) containing single nucleotide substitutions. We have developed an automated system for typing these simple biallelic markers that has several advantages over existing DNA typing methods. It is automated and has high throughput (1200 reactions/day) . It has a simple readout that is read directly by a computer. The reagents, oligonucleotides, are stable and safe (non-isotopic). The assay is easy to learn and relies on two widely used procedures, the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs). Furthermore , we find that clusters of linked biallelic pSTSs can be obtained with high heterozygosity (> 70%). Therefore, linked biallelic pSTSs can be as informative as manually typed simple or complex repeats. We will use biallelic pSTSs to construct a framework map of human chromosome 14 with an average marker spacing of 10 to 15 cM. We will obtain random pSTSs from genomic DNA samples using sequences from M13 libraries of flow-sorted human chromosome 14. These pSTSs will be rapidly and automatically ordered by genetic linkage analysis of informative CEPH families. Once a framework chromosome map is obtained, we will isolate YAC clones for each framework markers. This will produce physical anchors for the framework map and will also provide a source of sequences to obtain other pSTSs linked to each of the framework markers. Combined the linked biallelic pSTSs for each framework location will form an index marker (3 to 4 linked pSTSs) of high heterozygosity (> 70%). This will generate a framework map for human chromosome 14 that can be rapidly and automatically typed to detect genetic linkage of human disease-causing genes or phenotypic traits.
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