High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis?
详细信息 查看全文
- 作者：Lei Liu ; Vijaykumar Veerappan ; Qiaosheng Pu ; Chang Cheng ; Xiayan Wang ; Liping Lu ; Randy D. Allen ; Guangsheng Guo
- 刊名：Analytical Chemistry
- 出版年：2014
- 出版时间：January 7, 2014
- 年：2014
- 卷：86
- 期：1
- 页码：729-736
- 全文大小：436K
- ISSN：1520-6882
文摘
A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 渭m diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 脳 105 plates for the longest (48.5 kbp) and 3.7 脳 105 plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.