The detection of β-globin gene mutations in β-thalassemia using oligonucleotide probes and amplified DNA

Abstract

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in β-thalassemia. A series of oligonucleotide primers are synthesized which span the β-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the δ gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the β-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25–30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous β-thalassemia mutations.