Synthesis of highly modified DNA by a combination of PCR with alkyne-bearing triphosphates and click chemistry
Synthesis of highly modified DNA by a combination of PCR with alkyne-bearing triphosphates and click chemistry
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DOI：
10.1002/chem.200700502
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
--
作者：
Gierlich, Johannes;Gutsmiedl, Katrin;Carell, Thomas
我们报告的“点击化学”与PCR的组合，通过使用炔修饰的三磷酸盐的DNA的有效和均匀的标记。一系列不同长度（300，900和2000碱基对）的修饰的PCR产物的制备通过使用各种含炔和叠氮化物的三磷酸和不同的聚合酶。在实时PCR方法的密集筛选之后，开发了允许通过使用这些修饰的三磷酸以与标准PCR类似的效率扩增基因的方案。在高度修饰的PCR片段上的点击反应提供了90%以上的转化率，并导致在大DNA片段上的数百个炔的功能化，具有极好的选择性和效率。
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.