A PRACTICAL APPROACH TO HLA-DR GENOMIC TYPING BY HETERODUPLEX ANALYSIS AND A SELECTIVE CLEAVAGE AT POSITION-86

A common problem facing HLA-typing laboratories is to substitute genom ic typing for serology without having to handle a large number of olig oprobes, 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 th e procedure is the analysis of the DNA heteroduplexs, it shall be term ed HET typing. There are two stages: the first stage comprises two rou nds 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 latte r is designed with two nucleotide mismatches, thus creating an artific ial restriction site to differentiate between both HLA-DRB1 variants a t 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 res triction endonucleases. The digested or heteroduplexed samples are run on the same polyacrylamide gel. A total of 25 HLA-DRB1 alleles can th us be differentiated with a total of 10 primers and two restriction en zymes and without the use of probes. This protocol is ideally suited t o preliminary HLA class II typing of bone marrow donors.