Detection of the most common mutation of adenine phosphoribosyltransferase deficiency among Japanese by a non-radioactive method.

About 79% of all the Japanese patients with adenine phosphoribosyltransferase (APRT) deficiency have been estimated to possess at least one APRTJ allele with a substitution of ACG for ATG at codon 136. We developed a non-radioactive method for diagnosing genotypes of this disease. Part of the genomic DNA including the mutation site of the APRTJ allele was amplified using polymerase chain reaction and the amplified product was dot-blotted onto nylon membranes and then hybridized with either APRTJ-specific or non-APRTJ-specific synthetic oligonucleotides labelled at the 5' termini with biotin in the presence of non-labelled competitive synthetic sequences. The temperature was gradually decreased during the hybridization. When competitive sequences were omitted, difference in the intensity of the hybridization between APRTJ-containing and non-containing samples was not sufficiently clear to differentiate the genotypes. When an excess amount of competitive sequences was added in addition to biotin-labelled oligonucleotides, this method effectively differentiated samples containing only APRTJ alleles from those containing only non-APRTJ alleles. The present method was also useful to differentiate samples with both APRTJ and non-APRT*J alleles from those having only either of the alleles. An equivalent procedure using competitive sequence for hybridization and gradually decreasing the temperature will be useful for detecting point mutations in other genes.