COMPLETE HLA-A DNA TYPING USING THE PCR-RFLP METHOD COMBINED WITH ALLELE GROUP AND SEQUENCE-SPECIFIC AMPLIFICATION

We have established a practical method of complete high-resolution typ ing for all HLA-A alleles using the polymerase chain reaction (PCR)-re striction fragment-length polymorphism (RFLP) technique combined with allele group-and sequence-specific amplification, The second and third exons of the HLA-A gene, in which most allelic variations are observe d, were separately amplified by PCRs with 3 and 4 group-specific prime r pairs, respectively. Each PCR-amplified product was digested by alle le-specific restriction endonucleases and then subjected to electropho resis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A all eles could be discriminated by the RFLP patterns derived from the gene tic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HLA-A genotyping for all homozygous and hete rozygous combinations can be accomplished, establishing technically si mple, economical and practical routine typing of the HLA-A gene, espec ially for small samples.