ALLELE WALKING - A NEW AND HIGHLY ACCURATE APPROACH TO HLA-DRB1 TYPING - APPLICATION TO DRB1-ASTERISK-04 ALLELES

We have developed a typing method, which can be used even in small lab oratories, to produce a highly accurate and reliable allele assignment in any homozygous or heterozygous situation. We have called the metho d allele walking (AW) and it consists of sequential rounds of PCR-RFLP . After digestion, electrophoresis separates alleles positive for the mutation from the nega tive alleles; the cleaved fragment is then reco vered from the gel and analyzed for mutations at another codon. in thi s way, AW is able to positively ascertain which mutations are in the s ame chromosome (cis-linkage) and assigns alleles independently from ea ch other. Artificial sites are created in the PCR step in order to pos itively detect substitutions not naturally recognized by any of the ex isting or convenient enzymes. We report the application of AW for typi ng the 22 DRB104 alleles. The first PCR-RFLP round groups DRB1*04 all eles. Subsequently, the mutations at codons 86, 74, 71, 57 and 37 can be analyzed for the unambiguous assignment of the majority of the alle les. Additional polymorphisms at different codons can be assayed to re solve any undetermined alleles. The viability of all the restriction s ites used as well as the feasibility of AW were successfully tested.