Standard Reagents for Analyzing Human Genetic Diversity

A considerable number of DNBA restriction fragment length polymorphism (RFLPs) have beendiscovered and genotyped in populations and in reference families. RFPL's result from single base-pairdifferences within restriction enzyme recognition sequences which lead to fragments of variable length.Considerable population data has been assembled by "Southern analysis', a laborious procedure requiringmicrograms of DNA and hybridization with labeled probes. We have devised an innovative methodologyfor rapid conversion of RFPLs into a PCR-based format allowing reliable, cost-effective genotyping ofindividuals on a large scale. We will develop RFPL genotyping kits for approximately 200loci distributedthroughout the human genome as well as kits for rapid sequencing of any RFPL. Phase I includes:location and sequencing of approximately 500bp DNA domains containing variable restriction sites inplasmid, phage and cosmid clones, conversion from Southern to PCR analysis, development of aprototype kit for genotyping 3 RFPLs in a multiplex assay and applying it to 1 individual from each of19 populations, and international field testing. The genotyping kits are intended for 4 major users: (1)laboratories in the Human Genome Diversity project, (2) geneticists performing linkage analysis ofcomplex disorders, (3) epidemiologists pursuing disease association studies with molecular markers and(4) forensic investigators solving identification and paternity cases.