A practical approach to HLA-DR genomic typing by heteroduplex analysis and a selective cleavage at position 86

Abstract

A common problem facing HLA-typing laboratories is to substitute genomic typing for serology without having to handle a large number of oligoprobes, primers, or restriction enzymes. A protocol is described for HLA-DRB1 genomic typing using a combination of PCR amplification, DNA heteroduplex analysis, and restriction enzymes. Because the core of the procedure is the analysis of the DNA heteroduplexes, it shall be termed HET typing. There are two stages: the first stage comprises two rounds of PCR amplification of the polymorphic second exon of the HLA-DRB genes directly on lysed blood cells. The first amplification is with DRB generic primers, and the second amplification with seven HLA-DRB1 group-specific primers at the 5′ end and a common 3′ primer. The latter is designed with two nucleotide mismatches, thus creating an artificial restriction site to differentiate between both HLA-DRB1 variants at position 86, which is of critical importance in antigen presentation. The second stage involves subjecting the final amplified product to both DNA heteroduplex formation and digestion by two single-cutter restriction endonucleases. The digested or heteroduplexed samples are run on the same polyacrylamide gel. A total of 25 HLA-DRB1 alleles can thus be differentiated with a total of 10 primers and two restriction enzymes and without the use of probes. This protocol is ideally suited to preliminary HLA class II typing of bone marrow donors.