化学学报 ›› 2012, Vol. 70 ›› Issue (19): 2073-2078.DOI: 10.6023/A12030022 上一篇    下一篇

研究论文

电场方式对毛细管电泳分离DNA的影响

陈进a, 李振庆a,b, 倪一a, 刘晨晨a, 窦晓鸣a,b, 山口佳则a,c   

  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 上海交通大学物理系光学工程研究所 上海 200240;
    c 大阪大学先进光子研究中心 大阪 565-0871
  • 收稿日期:2012-05-18 出版日期:2012-10-14 发布日期:2012-06-21
  • 通讯作者: 李振庆 E-mail:zhenqingli@163.com
  • 基金资助:
    项目受国家自然科学基金(No. 21205078);上海市教委科研创新项目(No. 13YZ073);上海市科技发展基金(No. 10540500700)和上海理工大学教师创新能力建设(No. GDCX-Y-1205), 部分受上海市重点学科项目第三期(No. S30502)资助.

Effect of Electric Field Modes on the Separation Performance of DNA in Capillary Electrophoresis

Chen Jina, Li Zhenqinga,b, Ni Yia, Liu Chenchena, Dou Xiaominga,b, Yamaguchi Yoshinoria,c   

  1. a Engineering Research Center of Optical Instrument and System, Ministry of Education, Shanghai Key Lab of Modern Optical System, University of Shanghai for Science and Technology, Shanghai 200093;
    b Department of Physics, Optical Engineering, Shanghai Jiao Tong University, Shanghai 200240;
    c Photonics Advanced Research Center, Osaka University, Osaka 565-0871
  • Received:2012-05-18 Online:2012-10-14 Published:2012-06-21
  • Supported by:
    Project supported by the National Natural Science Foundation of China (No. 21205078), the Innovation Program of Shanghai Municipal Education Commission (No. 13YZ073), the Shanghai Committee of Science and Technology (China) (No. 10540500700), the Cultivating of Teacher’s Innovation Ability Program in University of Shanghai for Science and Technology (No. GDCX-Y-1205) and partly supported by the Leading Academic Discipline Project of Shanghai Municipal Government (No. S30502).

以羟乙基纤维素为筛分介质, 在直流、方波脉冲、反向脉冲电场中对0.1~10.0 kbp范围的DNA样品进行分离, 改变脉冲电场调制深度, 探讨电场方式对毛细管电泳分离DNA的影响. 研究发现, 其它实验条件一定时: (1)直流电场下, 小片段DNA (<1.0 kbp)可以被有效分离, 大片段DNA (>1.0 kbp)迁移时几乎重叠在一起; (2)方波脉冲电场下, 增大调制深度可提高大片段DNA (>1.0 kbp)分离效果, 但降低了部分小片段DNA (0.6~1.0 kbp)的分离度; (3)反向脉冲电场下, 可以实现0.1~8.0 kbp范围内各个DNA片段的有效分离, 改变调制深度会影响样本DNA的分离时间. 并将反向脉冲电场应用于毛细管电泳分离λ-DNA的EcoT14 I/Bgl II限制性内切酶酶切片段. 结果表明, 反向脉冲毛细管电泳技术具有快速、准确、重复性高等特点, 可用于宽分子量范围DNA片段分离.

关键词: 毛细管电泳, DNA, 羟乙基纤维素, 脉冲电场, 直流电场

DNA fragments ranging in size from 0.1 to 10.0 kbp were separated by constant electric field, square-wave pulsed electric field, and inversion pulsed electric field in hydroxyethylcellulose (HEC, 1300 K) polymer. The influence of electric field modes on the separation performance of DNA fragments was investigated when the modulation depth of pulsed electric field varied. We found that when other electrophoretic conditions were the same: (1) under constant electric field, DNA fragments can only be effectively resolved up to 1.0 kbp, whereas DNA fragments above 1.0 kbp co-migrated; (2) under square-wave pulsed electric field, high modulation depth favors the resolution of larger DNA fragments higher than 1.0 kbp, simultaneously the resolution of DNA fragments between 0.6 and 1.0 kbp deteriorates with the growth of modulation depth; (3) under inversion pulsed electric field, DNA fragments ranging in size from 0.1 and 8.0 kbp can be effectively resolved with high resolution. Furthermore, we have successfully separated the λ-DNA endonuclease fragments digested by EcoT14 I/Bgl II under inversion pulsed electric field. The results above demonstrate that inversion pulsed field capillary electrophoresis is a method with fast speed, accuracy, high reproducibility, and thus it can be applied for the separation of DNA fragments with a wide molecular range.

Key words: capillary electrophoresis, DNA, hydroxyethylcellulose, pulsed electric field, direct current electric field