COMPREHENSIVE METHOD FOR THE TYPING OF HLA-A, HLA-B, AND HLA-C ALLELES BY DIRECT SEQUENCING OF PCR PRODUCTS OBTAINED FROM GENOMIC DNA

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has signi ficant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine w hich alleles are present in a particular sample is to identify their s equence. We describe a simplified method for typing HLA-A, B, and C al leles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of s amples for clinical use. The template is the product of a nested PCR. A first round of PCR amplifications from genomic DNA is performed with three different sets of primers, one pair specific for each locus. Th e PCR products encompass exons 2 and 3, the regions of interest to det ermine the allele present. These fragments are a mixture of both allel es present in one locus. In a second round of PCRs using the first fra gment as template, exons 2 and 3 are separately amplified and simultan eously tailed with sequences corresponding to fluorescent-labeled comm ercial primers. The sense and antisense sequence of each exon is obtai ned and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles as signed. This simplified procedure has the practical advantage of allow ing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.