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

Shigeo Yoshida, Yoko Yamaji, Ayako Yoshida, Yasuhiro Ikeda, Ken Yamamoto, Tatsuro Ishibashi

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Purpose: Mutation 926delA of the arrestin/S-antigen SAG gene is the main cause of Oguchi disease in the Japanese. The purpose of this study was to develop a rapid diagnostic assay to detect mutations in the SAG gene. Methods: Two sequence-specific primers and fluorophore-labeled probes for exon 11 of the SAG gene were designed, and the region spanning 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 analyses of the hybrid formed between the PCR product and a specific fluorescent probe. Results: We clearly distinguished each SAG genotype (homozygous and heterozygous 926delA and wild type) by the distinct melting peaks at different temperatures. One thermal cycling required approximately 54 min to process, and the results were 100% in concordance with the genotypes determined by DNA sequencing. Conclusions: We have succeeded in developing a rapid method to detect the most frequent mutation in the SAG gene. This method will help in identifying gene mutations associated with Oguchi disease with a rapid and reliable identification or the exclusion of the frequent mutations in the SAG gene.

Original languageEnglish
Pages (from-to)1552-1557
Number of pages6
JournalMolecular vision
Volume12
Publication statusPublished - Dec 6 2006

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Real-Time Polymerase Chain Reaction
Mutation
Genes
Freezing
Genotype
Arrestin
Polymerase Chain Reaction
DNA Sequence Analysis
Fluorescent Dyes
Germany
Exons
Hot Temperature
Antigens
Temperature

All Science Journal Classification (ASJC) codes

  • Ophthalmology

Cite this

Yoshida, S., Yamaji, Y., Yoshida, A., Ikeda, Y., Yamamoto, K., & Ishibashi, T. (2006). Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction. Molecular vision, 12, 1552-1557.

Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction. / Yoshida, Shigeo; Yamaji, Yoko; Yoshida, Ayako; Ikeda, Yasuhiro; Yamamoto, Ken; Ishibashi, Tatsuro.

In: Molecular vision, Vol. 12, 06.12.2006, p. 1552-1557.

Research output: Contribution to journalArticle

Yoshida, S, Yamaji, Y, Yoshida, A, Ikeda, Y, Yamamoto, K & Ishibashi, T 2006, 'Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction', Molecular vision, vol. 12, pp. 1552-1557.
Yoshida S, Yamaji Y, Yoshida A, Ikeda Y, Yamamoto K, Ishibashi T. Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction. Molecular vision. 2006 Dec 6;12:1552-1557.
Yoshida, Shigeo ; Yamaji, Yoko ; Yoshida, Ayako ; Ikeda, Yasuhiro ; Yamamoto, Ken ; Ishibashi, Tatsuro. / Rapid detection of SAG 926delA mutation using real-time polymerase chain reaction. In: Molecular vision. 2006 ; Vol. 12. pp. 1552-1557.
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