title: Extensive screening system using suspension array technology to detect mitochondrial DNA point mutations.
abstract: We established an extensive and rapid system using suspension array to detect 61 representative mitochondrial DNA (mtDNA) heteroplasmic or homoplasmic point mutations (29 for Series A and 32 for Series B) in 22 genes: 1 each in MT-RNR1, -TV, -ND1, -TQ, -TW, -TC, and -TH genes; 2 each in MT-TN, -TG, -ND4, -TL2, -TE, and -CYB genes; 3 each in MT-ATP6, -ND3, and -ND5 genes; 4 each in MT-CO1 and -TK genes; 5 each in MT-TI, -TS1, and -ND6 genes; and 10 in the MT-TL1 gene. We carefully selected 5'-biotinylated primers and pooled primers for use in two sets of multiplex-PCR amplifications. To detect both mutant and wild-type mtDNA, even when polymorphisms were present near the target mutation sites, we designed specific oligonucleotide probes. By using the mtDNA point mutation detection system of Series A (29 mutations) and Series B (32 mutations), we screened a total of 3103 mutant sites in 107 DNA samples for Series A and 13,101 mutant sites in 397 DNA samples for Series B. We succeeded in determining 99.4% (Series A) and 99.6% (Series B) of the targeted mutant sites by use of the system. The 22 samples with the m.3243A>G heteroplasmic mutation revealed positive signals with both mutant- and wild-type-specific probes in this detection system with a detection limit of approximately 2%. This genetic screening platform is useful to reach a definitive diagnosis for mitochondrial diseases.
authors.affiliation: Department of Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan. yutakan@tmig.or.jp
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nishigaki
authors.forename: Hitomi
authors.initials: H
authors.lastname: Ueno
authors.forename: Jorida
authors.initials: J
authors.lastname: Coku
authors.forename: Yasutoshi
authors.initials: Y
authors.lastname: Koga
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujii
authors.forename: Ko
authors.initials: K
authors.lastname: Sahashi
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Nakano
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoneda
authors.forename: Michiko
authors.initials: M
authors.lastname: Nonaka
authors.forename: Linya
authors.initials: L
authors.lastname: Tang
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Liou
authors.forename: Veronique
authors.initials: V
authors.lastname: Paquis-Flucklinger
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Harigaya
authors.forename: Tohru
authors.initials: T
authors.lastname: Ibi
authors.forename: Yu-ichi
authors.initials: Y
authors.lastname: Goto
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hosoya
authors.forename: Salvatore
authors.initials: S
authors.lastname: DiMauro
authors.forename: Michio
authors.initials: M
authors.lastname: Hirano
authors.forename: Masashi
authors.initials: M
authors.lastname: Tanaka
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial