Rapid detection of SAG 926delA mutation using real-timepolymerase chain reaction

[摘要] Purpose: Mutation 926delA of the arrestin/S-antigen SAG gene isthe main cause of Oguchi disease in the Japanese. The purpose of thisstudy was to develop a rapid diagnostic assay to detect mutations in theSAG gene.Methods: Two sequence-specific primers and fluorophore-labeledprobes for exon 11 of the SAG gene were designed, and the regionspanning the mutations was amplified by polymerase chain reaction (PCR)using the LightCycler detection system (Roche Diagnostics, Mannheim,Germany). The mutations were then identified by melting curve analysesof the hybrid formed between the PCR product and a specific fluorescentprobe.Results: We clearly distinguished each SAG genotype (homozygousand heterozygous 926delA and wild type) by the distinct melting peaks atdifferent temperatures. One thermal cycling required approximately 54min to process, and the results were 100% in concordance with thegenotypes determined by DNA sequencing.Conclusions: We have succeeded in developing a rapid method todetect the most frequent mutation in the SAG gene. This method willhelp in identifying gene mutations associated with Oguchi disease with arapid and reliable identification or the exclusion of the frequentmutations in the SAG gene.

[学科分类] 生物化学/生物物理