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 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.
Jawaheer
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ARC Epidemiology Research Unit, University of Manchester, UK.
Ollier
W E
WE
Thomson
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D000483:Alleles; D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000276:immunology; D001327:Autoimmune Diseases / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D005838:Genotype; D015804:HLA-DR4 Antigen / Q000145:classification* / Q000235:genetics; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013379:Substrate Specificity; D013698:Templates, Genetic
D015804:HLA-DR4 Antigen; D015345:Oligonucleotide Probes