Abstract—

An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of “molecular beacon”-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or “click reaction” (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔC_t = 1–2.

中文翻译：

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带有荧光素双荧光团的分子信标 DNA 探针

摘要-

获得了荧光素双荧光团的叠氮衍生物，并用于合成“分子信标”型寡核苷酸荧光探针，用于 RT-PCR。基于优化的序列合成了八种探针变体：在 3' 端具有一个或两个猝灭剂残基，使用修饰的亚磷酰胺（短接头）或“点击反应”将单个或双荧光团荧光素标记连接到 5' 端。长链接器）。RT-PCR 中的探针比较表明，具有双重猝灭剂（​​短接头上的单一荧光素）和短接头上的双重染料（单一染料）的探针在灵敏度上比标准探针（单一猝灭剂，单一染料上的单一染料）要好一些。短链接器）由 ΔC _t = 1-2 的值决定。