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 (Hae II and/or Sac II) 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.