Research Institute of Physical-Chemical Medicine, Moscow, Russian Federation.
Bioconjug Chem. 2009 Aug 19;20(8):1441-3. doi: 10.1021/bc900076e. Epub 2009 Jul 14.
HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material with altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore, for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential, the careful RP-HPLC purification step is required.
通过典型的合成方案获得的 HEX 标记寡核苷酸可能含有超过 15%的光谱特性改变的物质。我们发现了以前未知的标准 DNA 氨解步骤中六氯荧光素残留的转化。HEX 残留与氨反应生成吖啶衍生物，与 HEX 相比，其具有不同的 UV-VIS 和荧光性质。因此，对于需要灵敏度和/或荧光信号区分（例如，在定量或多重测定中）的关键生物测定，需要进行仔细的反相高效液相色谱纯化步骤。
