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High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and
A R Gelsthorpe1, R S Wells, A P Lowe
1Cancer and Immunogenetics Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
Abstract:
We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.