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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.
Using hydroxyethylcellulose as screening medium, the DNA samples in the range of 0.1 ~ 10.0 kbp were separated in DC, square wave pulse and reverse pulse electric field, and the modulation depth of pulsed electric field was changed to discuss the effect of electric field on the separation of DNA by capillary electrophoresis (1) Small DNA fragments (<1.0 kbp) can be effectively separated under DC field and almost overlap when large DNA fragments (> 1.0 kbp) migrate; (2) Under the pulse electric field, increasing the modulation depth can improve the separation efficiency of large fragment DNA (> 1.0 kbp), but reduce the resolution of some small fragment DNA (0.6 ~ 1.0 kbp); (3) Under the reverse pulse electric field, The effective separation of DNA fragments in the range of 0.1 ~ 8.0 kbp can affect the DNA isolation time and the reverse pulse electric field can be applied to the EcoT14 I / Bgl II restriction endonuclease The results showed that reverse-phase pulse capillary electrophoresis was rapid, accurate and reproducible and could be used for DNA fragment separation in a wide range of molecular weight.