Grajkowski Andrzej, Cieślak Jacek, Beaucage Serge L
Laboratory of Biological Chemistry, Food and Drug Administration, Silver Spring, Maryland.
Curr Protoc Nucleic Acid Chem. 2017 Jun 19;69:10.17.1-10.17.30. doi: 10.1002/cpnc.31.
An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc.
本文介绍了一种高效纯化合成硫代磷酸酯和天然DNA序列的方法。该方法基于使用氨基丙基硅胶载体，其功能化有氨氧基烷基官能团，能够通过与连接到DNA序列5'端的连接子的酮官能团进行肟化反应来捕获DNA序列。作为5'-羟基保护基团，携带这种连接子的脱氧核苷亚磷酰胺已被合成，用于在可控孔径玻璃（CPG）载体上执行的标准固相合成方案的最后偶联步骤中掺入DNA序列。从CPG载体上释放的经碱基和磷酸去保护的DNA序列的固相捕获被证明几乎可以定量进行。首先将比全长DNA序列短的片段从捕获载体上洗去；然后，固相纯化的DNA序列在干燥的二甲基亚砜（DMSO）中与四丁基氟化铵反应后从该载体上释放出来，并沉淀在四氢呋喃（THF）中。固相纯化的DNA序列的纯度超过98%。展示了固相纯化过程的模拟高通量和可扩展性特征，且不影响DNA序列的纯度。© 2017 约翰威立父子公司版权所有
