Giller Gerald, Tasara Taurai, Angerer Bernhard, Mühlegger Klaus, Amacker Mario, Winter Holger
Gnothis SA, PSE-B, EPFL, CH-1015 Lausanne, Switzerland and Römerstrasse 7, D-82398 Polling, Germany.
Nucleic Acids Res. 2003 May 15;31(10):2630-5. doi: 10.1093/nar/gkg370.
Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. Vent(R) exo- DNA polymerase and a defined template-primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.
荧光标记的DNA是通过DNA聚合酶将荧光团连接的2'-脱氧核糖核苷-5'-三磷酸（dNTP）进行酶促掺入而产生的。我们描述了多种染料标记的dNTP的合成。所有四种天然存在的核碱基的氨基连接体修饰的5'-三磷酸被用作前体。市售染料与侧链的氨基功能基团偶联。此外，我们还连接了新型荧光团衍生物。经高效液相色谱（HPLC）纯化后，标记产物的纯度至少达到96%。研究了酶促掺入DNA以及随后修饰的DNA链的延伸情况。使用Vent(R) exo- DNA聚合酶和特定的模板-引物系统来分析每种染料标记的dNTP衍生物。我们的数据表明，掺入效率取决于所选的染料、核碱基或两者的组合。
