Prenatal diagnosis of beta-thalassemia. Detection of a single nucleotide mutation in DNA.

We investigated a method employing synthetic oligonucleotides for the prenatal diagnosis of beta-thalassemia due to a single nucleotide mutation. The beta 0 thalassemia we tested is produced by a nonsense mutation and is commonly found in Sardinia and other parts of the Mediterranean. In this DNA lesion, the glutamine codon CAG at the beta 39 position is mutated to TAG, which results in a stop codon and premature termination of the beta-globin chain. We synthesized two oligonucleotides: one homologous to the normal beta A gene and the other to the beta 0 thalassemia gene at the beta 39 location. The oligonucleotides were labeled with 32P and used as hybridization probes for normal and thalassemic DNA. The beta A probe hybridized only to the normal DNA, and the beta-thalassemia probe only to thalassemic DNA, thus providing a technique for direct demonstration of the mutation. The method is sensitive enough to be applied directly to DNA that is isolated from uncultured cells obtained from only 20 ml of amniotic fluid as early as the 16th gestational week.