HIGH THROUGHPUT GENOTYPING OF POPULATIONS.
          A. R. Gelsthorpe and J. G. Bodmer, Cancer Genetics and Immunology Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS .
          There is a current requirement in many laboratories for high throughput HLA genotyping system. Following our development of a semi-automated HLA class I typing system utilising TET/TAMRA labelled hydrolysis probes. We have integrated this system with a number of technologies to enhance the throughput of this system; which is suitable for either rapid tissue typing or for population studies. Reactions are carried out using either 96 or 384 well plates; the later are capable of up to four complete 4 typings (or 2 typings repeated) in 2 thermal cyclers with four heads apiece. We use a rapid cycling protocol which completes 35 cycles in under 45 minutes. Reagents are plated using a combination of a liquid handling robot in conjunction with a 96 well dispenser. Detection of the cleaved probes is achieved with a BMG FLUOstar Scanner. A 96 well plate can be scanned in under 30 seconds. When all these technologies are integrated the system is capable of a complete typing in under 1 hour, or if all the thermal cyclers are fully utilised 14 typings per hour. The results from 200 plus samples are in full accordance with conventional gel based systems. Further enhancement of the system is being investigated with previously described magnetic beads coated with anti DNA monoclonal antibody; these are capable of preparing template from either blood or soft tissue in under 30 minutes. Following the use of dyes such as YO-PRO and SYBR Green we are testing a new generation of intercalating DNA dyes which are incorporated into the reaction chemistry pre-PCR, but which have significantly lower affinity for single stranded DNA than current reagents. This results in a very low background signal and facilitates the separation of positive from negative reactions. To conclude; we have developed a semi-automated genotyping system, with a throughput greatly in excess of current approaches.