1998 Volume 5 Issue 1 Pages 4-17
We developed a nested polymerase chain reaction (PCR) -restriction fragment length polymorphism (RFLP) method for high-resolution typing of the HLA-B alleles. HLA-B alleles could be identified by this method without the need of other information such as serological type. The first PCR was performed using HLA-B locus specific primers to obtain a 940 bp DNA fragment covering from exon 1 through exon 3. In the second PCRs, exon 2 fragments of HLA-B alleles were amplified from the diluted first PCR product using 2 sets of group specific primers, and exon 3 fragments were amplified using 3 sets of group specific primers independently. Computer analysis of cleavage patterns for 178 HLA-B alleles showed that 60 RFLP patterns could be obtained by digestion of the exon 2 PCR products and 80 RFLP patterns by digestion of the exon 3 PCR products. Based on the combinations of both exons patterns, 127 individual alleles could be identified, remaining 5 1 alleles could be classified into 19 allele-groups each of which includes 2 to 4 alleles. We could exactly identify 46 HLA-B alleles from 56 cell lines of the UCLA International Cell Exchange Program using the present PCR-RFLP method. This method is useful for the high-resolution typing of HLA-B alleles for a relatively small number of samples.
