PCR-RFLP analysis of the IVS3 + 5 G → A splice site mutation in ABO genes.
The IVS3 + 5G → A mutation identified in the B gene of the B3 propositus creates a BsmI site. The 247-bp PCR product, encompassing the exon 3–intron 3 junction, amplified from the gene with the IVS3 + 5G → A mutation yielded 206- and 41-bp fragments, after digestion with BsmI restriction endonuclease, while the 247-bp PCR product from a wild-type gene was resistant to digestion. The BsmI-cleaved products were analyzed by 3.5% agarose gel electrophoresis. Samples from the B3 propositus (lane 1) and another 13 unrelated B3 individuals (lanes 2-14) together with samples from 30 randomly selected group B individuals were subjected to PCR-RFLP analysis. One of the results obtained from the group B individuals is shown in lane B. Lane M shows the molecular mass standards of the 100-bp ladder. The results indicate that 13 of the 14 B3individuals, all but the 1 shown in lane 14, possessed one allele with the IVS3 + 5G → A mutation, while none of the 30 group B individuals had the mutation.
