MULTIPLEX ARMS-RFLP - A SIMPLE AND RAPID METHOD FOR HLA-DR4 SUBTYPING

We have developed a simple and rapid non-radioactive technique for HLA -DR4 subtyping. A multiplex ARMS-RFLP (Amplification Refractory Mutati on System -Restriction Fragment Length Polymorphism) system allows HLA -DR4 subtyping by analysis of the products of two multiplex ARMS react ion mixtures. For some cases, restriction enzyme digests (Hae II and/o r Sac II) of the products are analysed. The technique relies on the fa ct that an ARMS primer with a mismatch at its 3'-end with respect to t he template will not be elongated under PCR conditions. Hence, by desi gning ARMS primers such that different HLA-DR4 alleles yield PCR produ cts of different lengths, only two reactions, each using a mixture of different ARMS primers, are sufficient to type all of the known HLA-DR 4 alleles. This system can distinguish between HLA-DR4 'homozygotes' a nd 'heterozygotes' since every HLA-DR4 allele can be detected. The ARM S conditions were optimized using DNA from cell lines. This technique has now been used to type a panel of rheumatoid arthritis patients and controls.