Detection of the most common mutations causing beta-thalassemia in Mediterraneans using a multiplex amplification...

A rapid, simple, cost-effective, non-radioactive method for detection of the most common mutations causing beta-thalassemia in Mediterranean people has been developed by combining multiplexing with the amplification refractory system. This approach, the multiplex amplification refractory mutation system (MARMS), provides an easy assay for direct detection of normal and mutant beta-globin genes in homozygotes and heterozygotes. The strategy involves multiplex PCR of four of the five regions of interest within the beta-globin gene in a single reaction containing a common oligoprimer and either the normal or mutant oligonucleotides corresponding to IVS-1 nucleotide 1 or IVS-1 nucleotide 6, IVS-1 nucleotide 110, codon 39, and IVS-2 nucleotide 1 regions. Primers are chosen so that the sizes of the four PCR products differ, thereby facilitating detection on agarose gels following amplification. Patient samples are primed with either four normal or four mutant oligonucleotide mixtures and the common oligoprimer, and PCR products run in parallel on gels to detect band presence or absence. This approach simplifies mutation detection and shows promise for automation employing fluorescent-tagged primers.