A simple “add and measure” FRET-based telomeric tandem repeat sequence detection and telomerase assay method
A simple “add and measure” FRET-based telomeric tandem repeat sequence detection and telomerase assay method
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一种简单的“添加和测量”基于 FRET 的端粒串联重复序列检测和端粒酶测定方法
DOI：
10.1039/c3ob42092b
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Takashi Murashima
中科院分区：
文献类型：
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作者：
Koji Kawamura;Hidenobu Yaku;Daisuke Miyoshi;Takashi Murashima
以FAM修饰的12-mer ODN探针为供体（荧光团），溴化乙锭（EB）为受体（猝灭剂），建立了一种简单、灵敏的基于荧光共振能量转移（FRET）的端粒串联重复序列DNA和端粒酶活性检测方法。当端粒DNA和FAM修饰的探针形成双链体时，EB插入碱基对之间，导致FAM通过从FAM到EB的FRET的荧光猝灭。该方法可用于估计样品溶液中端粒DNA的量，作为端粒DNA单元的摩尔浓度[5′-（TTA GGG TTA GGG）-3′]。通过调节FAM修饰的探针的量，在5-1000 pM的端粒DNA单元中获得线性荧光猝灭比。该方法无需PCR检测，可检测≥400个HeLa细胞/μL。该测定代表了用于癌症诊断的初始筛选的新技术，并且是用于定量端粒DNA或其他串联重复序列的简便方法。
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.