化学学报 ›› 2013, Vol. 71 ›› Issue (02): 265-270.DOI: 10.6023/A12110935 上一篇    下一篇

研究论文

羟乙基纤维素中毛细管电泳DNA分离特性研究

刘晨晨a, 李振庆a, 孟凡明a, 倪一a, 窦晓鸣a, 山口佳则b,a   

  1. a 教育部光学仪器与系统工程研究中心 上海市现代光学系统重点实验室 上海理工大学光电信息与计算机工程学院 上海 200093;
    b 大阪大学先进光子学中心 大阪 565-0871
  • 收稿日期:2012-11-19 出版日期:2013-02-14 发布日期:2012-12-25
  • 通讯作者: 李振庆 E-mail:zhenqingli@163.com
  • 基金资助:

    国家自然科学基金(No. 21205078);高等学校博士学科点专项科研基金博导类资助课题(No. 20123120110002);上海理工大学教师创新能力建设(No.GDCX-Y-1205);上海市教委科研创新项目(No. 13YZ073);上海市科技发展基金(No. 10540500700)和上海市研究生创新基金(No. JWCXSL1202)资助项目. 本文部分受上海市重点学科项目第三期项目(S30502)资助.

Capillary Electrophoresis of DNA in Hydroxyethylcellulose

Liu Chenchena, Li Zhenqinga, Meng Fanminga, Ni Yia, Dou Xiaominga, Yamaguchi Yoshinorib,a   

  1. a Engineering Research Center of Optical Instruments and System, Ministry of Education, Shanghai Key Laboratory of Modern Optical System, School of Optical-Electrical and Computer Engineering, University of Shanghai for Science and Technology, Shanghai 200093;
    b Photonics Advanced Research Center, Osaka University, Osaka 565-0871
  • Received:2012-11-19 Online:2013-02-14 Published:2012-12-25
  • Supported by:

    Project supported by the National Natural Science Foundation of China (No. 21205078), Research Fund for the Doctoral Program of Higher Education of China (No. 20123120110002), Teachers’ Innovation Ability Construction of University of Shanghai for Science and Technology (No. GDCX-Y-1205), the Innovation Program of Shanghai Municipal Education Commission (No. 13YZ073), Shanghai Committee of Science and Technology (No. 10540500700) and the Innovation Fund Project For Graduate Student of Shanghai (No. JWCXSL1202), and partly supported by the Leading Academic Discipline Project of Shanghai Municipal Government (S30502).

本实验以羟乙基纤维素(HEC)为筛分介质, 以100~1500 bp DNA ladder 为分离对象, 系统地研究了直流电场下毛细管电泳时DNA 分离特性. 论文考察了DNA 迁移淌度及分离度随HEC 溶液浓度和分子量、毛细管两端电场强度(E)、毛细管有效长度(le)及其内径形状、背景电解液(BGE)温度等因素变化规律. 研究发现: (1)当筛分介质HEC 浓度高于其阈值浓度c*时, HEC 分子量越大, 相邻DNA 片段之间淌度差越大, HEC 浓度越高, 其迁移淌度越低; (2)对于相邻的DNA 片段, le在一定范围内, 其分离度随le增大而线性升高; (3)毛细管有效长度一定时, DNA 淌度随毛细管侧面积与截面积之比R增大而升高, 分离效率提高; (4) BGE 温度升高, DNA 在筛分介质中扩散效应增强, 迁移淌度变大, 相邻DNA片段间分离度减小. 根据以上结论, 在直流电场下毛细管电泳φ×174-Hirc II 限制性酶切片段, 并实现了其高分离度、快速分离.

关键词: 毛细管电泳, 羟乙基纤维素, DNA

Using hydroxyethylcellulose (HEC) solution as polymer matrix, this work systematically studied the separation performance of 100 base pairs (bp) DNA ladder (100~1500 bp) by direct current electric field capillary electrophoresis (CE). In the present paper, we systematically investigated the influence of polymer concentration and molecular weight of HEC, electric field strength (E), the effective length (le) and the shape of the capillary, the temperature of the background electrolyte (BGE) on the separation performance of DNA. Furthermore, we compared the migration of DNA in polymer with the non-gel sieving model. Results show that: (1) When the concentration of HEC is above the entangled threshold c*, the mobility difference increases with the growth of molecular weight, whereas the mobility of DNA decreases with the rise of concentration of HEC. (2) The resolution of adjacent DNA fragments linearly increases with the effective length (le) of the capillary when le ranges from 4 to 12 cm. (3) The mobility of DNA increases with the growth of area ratio R (Slateral/Ssection), and thus the separation performance improves. (4) The increase of BGE temperature strengthens the diffusion effect of DNA, thus increases the mobility, and deteriorates the resolution. Based on the results above, we separated the φ×174-Hirc II digest by CE in an optimal electrophoretic condition. Experiment shows that rapid separation of φ×174-Hirc II digest was realized with high resolution. In our experiment, the fused-silica capillary is coated by acrylamide, and the background electrolyte (BGE) used for the sieving matrix contains 0.5×Tris-borate-EDTA (TBE) and 1×SYBR Green I. The DNA sample was injected for 2.0 s at an E of 100 V/cm. The self-build CE device involved is reliable and showed some remarkable achievements previously. Such a study is beneficial to the realization of rapid and effective separation of DNA, and allows deep insight into DNA migration in the polymer matrix under constant electric field.

Key words: capillary electrophoresis, hydroxyethylcellulose, DNA