Dias Rita S, Svingen Roine, Gustavsson Bodil, Lindman Björn, Miguel Maria G, Akerman Björn
Chemistry Department, University of Coimbra, Portugal.
Electrophoresis. 2005 Aug;26(15):2908-17. doi: 10.1002/elps.200400182.
We use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coil size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications.
我们使用琼脂糖凝胶电泳来表征单价阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）如何使双链DNA凝聚，这可通过DNA电泳速度的降低来检测。当CTAB与DNA磷酸基团电荷的比例R = 1.8时，即高于中和点时，速度达到平稳状态，并且复合物至少在R = 200之前都保持净负电荷。对两种不同大小DNA混合物的凝聚实验表明，形成的复合物各自仅包含一个凝聚的DNA分子。通过对凝胶内其速度的时间分辨测量对这些CTAB-DNA小球进行了进一步表征，结果表明CTAB在迁移过程中不会解离，但可能在进入凝胶时解离。使用奥格斯顿球形颗粒电泳模型，测得的小球在凝胶中的速度在定量上与CTAB具有两种相反作用一致，即降低电泳电荷和减小DNA线圈大小。就CTAB而言，这两种作用几乎相互抵消，这可以解释为什么使用某些阳离子凝聚剂时会观察到相反的速度变化（小球比未复合的DNA快）。还研究了通过添加阴离子表面活性剂使复合物解离的情况。在阴离子和阳离子表面活性剂的混合比例等于1时，DNA从小球中完全释放，这与平衡研究结果一致。环状DNA保留了其超螺旋结构，这表明在压实-释放循环中不存在DNA切口，这在DNA转染和纯化应用中很重要。
