Yu Shengbing, Geng Jing, Zhou Ping, Wang Jing, Feng Airong, Chen Xiangdong, Tong Hua, Hu Jiming
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
Anal Chim Acta. 2008 Mar 24;611(2):173-81. doi: 10.1016/j.aca.2008.01.050. Epub 2008 Feb 2.
A new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid (DNA) extraction was prepared in situ by polymerization of N-(beta-aminoethyl)-gamma-aminopropyltriethoxysilane (AEAPTES) and tetraethoxysilane (TEOS). The main extraction mechanism was based on the Coulombic force between DNA and the amino silica hybrid monolithic column. DNA extraction conditions, such as pH, ion concentration and type, and loading capacity, were optimized online by capillary electrophoresis with laser-induced fluorescence detection. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 48 ng DNA with an extraction efficiency of 74+/-6.3% (X+/-RSD). The DNA extraction process on this monolithic column was carried out in a totally aqueous system for the successful purification of DNA and removal of proteins. The PBE2 plasmid could be extracted from Bacillus subtilis (B. subtilis) crude lysate within 25 min, and the purified DNA was suitable for the amplification of a target fragment by polymerase chain reaction. This study demonstrates a new attractive solid-phase support for DNA extraction to meet the increasingly miniaturized and automated trends of genetic analyses.
通过N-(β-氨乙基)-γ-氨丙基三乙氧基硅烷（AEAPTES）和四乙氧基硅烷（TEOS）原位聚合制备了一种用于高效脱氧核糖核酸（DNA）提取的新型有机-无机混合整体柱。主要提取机制基于DNA与氨基硅胶混合整体柱之间的库仑力。采用激光诱导荧光检测的毛细管电泳在线优化了DNA提取条件，如pH值、离子浓度和类型以及负载量。在最佳条件下，一根6.0厘米的整体柱可容纳48纳克DNA，提取效率为74±6.3%（X±RSD）。该整体柱上的DNA提取过程在完全水相体系中进行，以成功纯化DNA并去除蛋白质。PBE2质粒可在25分钟内从枯草芽孢杆菌粗裂解物中提取出来，纯化后的DNA适用于通过聚合酶链反应扩增目标片段。本研究展示了一种用于DNA提取的新型有吸引力的固相支持物，以满足基因分析日益小型化和自动化的趋势。
