Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays
Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays
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DOI：
10.1021/ja043595x
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发表时间：
2005-03-02
影响因子：
15
通讯作者：
Fuller, CW
中科院分区：
文献类型：
--
作者：
Sood, A;Kumar, S;Fuller, CW
对于DNA和RNA聚合酶来说，具有染料的核苷酸与具有四个或更多个磷酸（四磷酸或五磷酸）的末端磷酸连接，是比相应的三磷酸更好的上级底物。当荧光染料直接连接到末端磷酸盐时，它们可以通过聚合酶和碱性磷酸酶的作用释放。释放的染料改变颜色和荧光特性。也可以通过使用多个标记的核苷酸来扩增荧光信号以检测少量的模板。我们已经探索了这些核苷酸在各种应用中的效用，包括同质SNP检测方法、DNA测序以及PCR和RCA的定量。
Nucleotides with a dye attached to the terminal phosphate with four or more phosphates (tetra- or pentaphosphates) are superior substrates than the corresponding triphosphates for DNA and RNA polymerases. When fluorogenic dyes are directly attached to the terminal phosphate, they can be released by the action of polymerase and alkaline phosphatase. The released dye changes color and fluorescence properties. The fluorescent signal can also be amplified by using multiple labeled nucleotides to detect small amounts of template. We have explored the utility of these nucleotides in a variety of applications including homogeneous SNP detection methods, DNA sequencing, and quantitation of PCR and RCA.