Comprehensive method for the typing of HLA-A, B, and C alleles by direct sequencing of PCR products obtained from genomic DNA.

Abstract

Molecular testing is gradually replacing standard typing techniques in the field of HLA because it allows higher resolution, which has significant functional implications. Although several techniques have been so far described for this purpose, the definitive means to determine which alleles are present in a particular sample is to identify their sequence. We describe a simplified method for typing HLA-A, B, and C alleles by direct sequencing of polymerase chain reaction (PCR) products amplified from genomic DNA that could allow large-scale handling of samples 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. The PCR products encompass exons 2 and 3, the regions of interest to determine the allele present. These fragments are a mixture of both alleles present in one locus. In a second round of PCRs using the first fragment as template, exons 2 and 3 are separately amplified and simultaneously tailed with sequences corresponding to fluorescent-labeled commercial primers. The sense and antisense sequence of each exon is obtained and compared with a database of all known HLA-A, B, or C alleles. Heterozygous positions are determined and the most probable alleles assigned. This simplified procedure has the practical advantage of allowing high-resolution typing of clinical material by utilizing the same genomic DNA used for standard molecular typing of HLA class I.