Andrzej Grajkowski, Jacek Cieślak, Serge L. Beaucage
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引用次数: 6
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Abstract
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.
合成DNA序列固相纯化的高通量工艺
提出了一种高效的合成硫代磷酸酯和天然DNA序列的纯化方法。该工艺是基于使用氨基基烷基功能化的氨丙化硅胶载体,通过与DNA序列5 '端共轭的连接器的酮功能的氧化反应来捕获DNA序列。携带该连接体的脱氧核糖核苷磷酰胺作为5 ' -羟基保护基团,在受控孔玻璃(CPG)载体上的标准固相合成程序的最后一步被合成并整合到DNA序列中。从CPG载体中释放的核碱基和磷酸盐去保护DNA序列的固相捕获被证明是接近定量的。短于全长的DNA序列首先从捕获支架上冲洗掉;固相纯化的DNA序列在干燥的二甲基亚砜(DMSO)中与四正丁基氟化铵反应后从该载体中释放出来,并在四氢呋喃(THF)中沉淀。固相纯化的DNA序列纯度超过98%。在不牺牲DNA序列纯度的情况下,模拟了固相纯化过程的高通量和可扩展性特征。©2017 by John Wiley &儿子,Inc。
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