High-throughput class IHLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR)

We have developed a semi-automated HLA class I typing system utilising TET/ TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis pro bes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive co nventional gel-based DNA typing and has a higher allelic resolution than se rology. Our approach differs from previously published fluorogenic probe ty ping protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not spec ific to FRET probe analysis our system has in-built expansion capacity to 3 84 reactions per plate, thus making it applicable to high-throughput popula tion screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high th roughput with a low inherent error rate.