SELECTION OF UNRELATED BONE-MARROW DONORS BY PCR-SSP TYPING AND SUBSEQUENT NONRADIOACTIVE SEQUENCE-BASED TYPING FOR HLA DRB1 3/4/5, DQB1, AND DPB1 ALLELES/

HLA incompatibility between bone marrow recipients and unrelated donor s is one of the main obstacles in bone marrow transplantation. HLA cla ss I and generic class II DR and DQ typing is generally performed by s erology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Hen, the final selection of se rologically pretyped unrelated bone marrow donors by confirmatory PCR- SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marr ow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separat ion, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophor-labelled sequencing prim ers. Electrophoretically separated sequencing products were detected b y means of an automated DNA sequencer. Group-specific amplification an d sequencing of DQB1 alleles was carried out for all potential bone ma rrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strat egy in combination with direct sequencing of PCR products allows match ing of bone marrow transplant pairs with the highest degree of reliabi lity for the assessment of HLA class II identity.