A 3-STEP ALLELE-LEVEL DRB1-DRB3-DRB4-DRB5 GENOTYPING ASSAY USING POLYMERASE CHAIN-REACTION WITH IMMOBILIZED SEQUENCE-SPECIFIC OLIGOPROBES

A three-step reverse hybridization assay for allele level-resolution D RB1-DRB3-DRB4-DRB5 genotyping is described. Samples are initially ampl ified using a generic primer pair for all DRB1-DRB3-DRB4-DRB5 alleles and PCR products are hybridized to generic typing membranes. An interm ediate-resolution level genotyping is obtained at this point. Dependin g on the phenotype, samples are then subjected to a DR1, DR2, DR4, DR5 2A, DRB3 and/or DRB5 type-specific amplification and hybridization. A third step, involving sequence-specific PCR followed by type-specific hybridization, is only performed to solve certain DR4 and DR52A hetero -zygous combinations. The assay allows 100% allele level-resolution DR B genotyping. Hybridization membranes contain panels of SSO probes tha t were optimized to all react specifically under identical stringency conditions. A computer program was written to assist in analysis of th e hybridization patterns. The assay was thoroughly evaluated and has b een used to type over 10,000 donors from the Canadian Unrelated Bone M arrow Donor Registry (UBMDR) at allele level-resolution. This method p roved to be flexible, easy to update for newly described alleles, easy to perform, fast, and safe. It is also reliable and specific, as 9 no vel DRB alleles so far have been detected as aberrant hybridization pa tterns.