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 Mutation System Restriction Fragment Length Polymorphism) system allows HLA-DR4 subtyping by analysis of the products of two multiplex ARMS reaction mixtures. For some cases, restriction enzyme digests (HaeII and/or SacII) of the products are analysed. The technique relies on the fact that an ARMS primer with a mismatch at its 3'-end with respect to the template will not be elongated under PCR conditions. Hence, by designing ARMS primers such that different HLA-DR4 alleles yield PCR products of different lengths, only two reactions, each using a mixture of different ARMS primers, are sufficient to type all of the known HLA-DR4 alleles. This system can distinguish between HLA-DR4 'homozygotes' and 'heterozygotes' since every HLA-DR4 allele can be detected. The ARMS conditions were optimized using DNA from cell lines. This technique has now been used to type a panel of rheumatoid arthritis patients and controls.