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

1744-313X

Blackwell Publishing Journal Backfiles 1879-2005

Biology

Medicine

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.

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