A simple "add and measure" FRET-based telomeric tandem repeat sequence detection and telomerase assay method

作者：Kawamura Koji; Yaku Hidenobu; Miyoshi Daisuke; Murashima Takashi^*

来源：Organic and Biomolecular Chemistry, 2014, 12(6): 936-941.

DOI：10.1039/c3ob42092b

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 mu L. 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.

• 出版日期2014