The aim of this proposal is to increase the throughput of a single denaturing HPLC instrument so that on a single day 12,000 bp can be surveyed for simple sequence variation in 96 individuals, amounting to a total of 1.15Mb screened. Denaturing HPLC (DHPLC) has proven to be a sensitive and cost-effective method for detecting simple sequence polymorphisms. DHPLC exploits the decrease in retention caused by mismatched base pairs. The magnitude of this decrease can be predicted, hence eliminating the need for determining the optimum temperature of analysis experimentally. Since different mismatches cause usually different heteroduplex profiles only one representative individual per unique profile needs to be sequenced. This will reduce the number of conventional sequencing runs required per screened fragment to approximately four out of 96 individuals surveyed. Furthermore, as the screening set of 96 DNA samples will remain unaltered, further reduction in cost and streamlining of the laboratory protocol can be attained by the use of a 96-capillary thermocycling device that also functions as a liquid handling robot. This will eliminate such consumables as tubes and pipette tips, and due to the reduced reaction volume, the amount of Taq polymerase required for amplification. Recent progress made in the identification of population specific haplotypes both on autosomes as well as the Y chromosome will provide the rationale for selecting individuals to be included in the final screening set of 96 individuals. The same information will allow the pooling of chromosomes. Since coding and regulatory sequences constitute less than 3% of the primary sequence generated within the context of the human genome effort, less than 20 instruments will suffice to assess common variation at an annual primary sequencing production rate of 500 Mb.
