A rapid and reliable method for direct genotyping of codon 360 in the human apolipoprotein A-IV gene

Human apolipoprotein A-IV exhibits a polymorphism, first investigated at the protein level, that is caused by a single amino acid substitution of glutamine to histidine at codon 360. Detection of this polymorphism requires polymerase chain reaction (PCR) and direct sequencing of the amplified products, radiolabeled allele-specific oligonucleotides (ASOs) technique, or restriction enzyme analysis of the amplified products. However, these techniques involve the use of radioactivity and/or are not well suited to the rapid processing of a large number of samples. In this paper, we propose a new technique, a bispecific-allele primer amplification, in which a simple electrophoresis of PCR products is used for typing the variation at codon 360. The 3' primer of PCR hybridizes with one or other homologous sequence in the apoA-IV gene, depending on the presence or the absence of the mutation. This differential hybridization of the primer is used for typing the variation. In order to demonstrate the validity of this system, 120 individuals phenotyped by two-dimensional electrophoresis and genotyped by ASO were analyzed by this new technique. The results obtained by the latter method are in agreement with those found by the other techniques. However, this method is simple, more reliable, and will facilitate population studies without using radioactive materials.