Gierlich Johannes, Gutsmiedl Katrin, Gramlich Philipp M E, Schmidt Alexandra, Burley Glenn A, Carell Thomas
Department of Chemistry and Biochemistry, Ludwig-Maximilians University Munich, Germany.
Chemistry. 2007;13(34):9486-94. doi: 10.1002/chem.200700502.
We report the combination of "click chemistry" with PCR by using alkyne-modified triphosphates for efficient and homogeneous labeling of DNA. A series of modified PCR products of different lengths (300, 900, and 2000 base pairs) were prepared by using a variety of alkyne- and azide-containing triphosphates and different polymerases. After intensive screening of real-time PCR methods, protocols were developed that allow the amplification of genes by using these modified triphosphates with similar efficiency to that of standard PCR. The click reaction on the highly modified PCR fragments provided conversion rates above 90 % and resulted in the functionalization of hundreds of alkynes on large DNA fragments with superb selectivity and efficiency.
我们报告了通过使用炔烃修饰的三磷酸酯将“点击化学”与聚合酶链式反应（PCR）相结合，以实现对DNA的高效且均一的标记。使用多种含炔烃和叠氮化物的三磷酸酯以及不同的聚合酶，制备了一系列不同长度（300、900和2000个碱基对）的修饰PCR产物。在对实时PCR方法进行深入筛选后，开发出了一些方案，这些方案允许使用这些修饰的三磷酸酯扩增基因，其效率与标准PCR相似。在高度修饰的PCR片段上进行的点击反应提供了高于90%的转化率，并以极高的选择性和效率实现了大DNA片段上数百个炔烃的功能化。
