RAPID DNA TYPING OF CLASS-II HLA ANTIGENS USING THE POLYMERASE CHAIN-REACTION AND REVERSE DOT BLOT HYBRIDIZATION

A nonisotopic oligotyping method using reverse dot blot hybridization was developed for HLA class II DQA1, DQB1, DPB1, DRB1, DRB3, DRB4, DRB5 alleles. The polymorphic second exon of the different genes was amplified by the polymerase chain reaction (PCR). For each gene the amplified DNA was hybridized at stringent conditions to membrane-bound sequence-specific oligonucleotides (SSOs) and visualization of positive signals was done by chemiluminescence. A combination of 11, 18, 23 and 31 SSOs was designed to identify 9/13 DQA1, 16/17 DQB1, 23/24 DPB1 and 50/55 DRB1, 4 DRB3, 1 DRB4,3/4 DRB5 alleles respectively. For the DRB1 locus, an additional DRB1*04 group-specific PCR was developed to make discrimination between the DR4 alleles possible in different heterozygous combinations. The procedure described here provides rapid and nonisotopic genotyping of heterozygous samples from a variety of sources and can be applied for tissue typing, disease susceptibility studies and forensic medicine.