Polymerase chain reaction (PCR) mutagenesis enabling rapid non-radioactive detection of common beta-thalassaemia mutations in Mediterraneans.

The IVS-1-110 (G----A) and IVS-1-1 (G----A) mutations occur in approximately 33% and 9% respectively of beta-thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)-amplified material by allele-specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3' base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS-1-110) and HinfI (IVS-1-1) produced different fragments on electrophoresis. The above strategy was validated by allele-specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS-1-110, codon 39 and IVS-1-1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.