电场方式对毛细管电泳分离DNA的影响

摘要

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0．1～10．0kbp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（〈1．0kbp）可以被有效分离，大片段DNA（〉1．0kbp）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（〉1．0kbp）分离效果，但降低了部分小片段DNA（0．6～1．0kbp）的分离度；（3）反向脉冲电场下，可以实现0．1～8．0kbp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EcoTl4I／BglII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．%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 re- solved with high resolution. Furthermore, we have successfully separated the 2-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.