title: A simple "add and measure" FRET-based telomeric tandem repeat sequence detection and telomerase assay method.
abstract: A simple and sensitive method for measuring telomeric tandem repeat DNA and telomerase activity based on fluorescence resonance energy transfer (FRET) with a FAM-modified 12-mer ODN probe as a donor (fluorophore) and ethidium bromide (EB) as an acceptor (quencher) is proposed. When telomeric DNA and the FAM-modified probe form a duplex, EB intercalates between base-pairs, resulting in fluorescence quenching of FAM through FRET from FAM to EB. This method can be used to estimate the amount of telomeric DNAs in a sample solution as the molar concentration of the telomeric DNA unit [5'-(TTA GGG TTA GGG)-3']. A linear fluorescence quenching ratio was obtained in 5-1000 pM of telomeric DNA units by adjusting the amount of FAM-modified probe. A PCR-free telomerase activity assay using this FRET-based method could be applied to ≥400 HeLa cells per μL. This assay represents a novel technique for initial screenings of cancer diagnosis and is a facile method for quantifying telomeric DNA or other tandem repeat sequences.
authors.affiliation: Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. murasima@konan-u.ac.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Kawamura
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Yaku
authors.forename: Daisuke
authors.initials: D
authors.lastname: Miyoshi
authors.forename: Takashi
authors.initials: T
authors.lastname: Murashima
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004247:DNA; D019098:Telomerase