Bünemann H
Nucleic Acids Res. 1982 Nov 25;10(22):7181-96. doi: 10.1093/nar/10.22.7181.
The accessibility of immobilized DNA has been shown to depend more crucially on the method of immobilization than on the type of support used for fixation. When sonicated denatured DNA is coupled via diazotization or via cyanogen bromide reaction to solid Sephadex G-25 and Cellex 410 or to macroporous Sephacryl S-500 and Sepharose C1-6B its accessibility varies from 100 to 24 percent. Generally the loss of accessibility is linked to a depression of the melting temperature of DNA helices formed on the support. This correlation shows a characteristic course for a particular coupling method. DNA coupled under denaturing conditions may become totally inaccessible when only 3 percent of its bases are involved in the covalent linkage. Kinetic experiments with sonicated E.coli DNA have shown that the rate constants for renaturation or hybridization reactions are very similar for DNA immobilized by different methods to solid or macroporous supports. Generally the second order rate constant for a heterogeneous reaction (between mobile and immobilized DNA) is about one order of magnitude smaller than that of the analogous homogeneous reaction (in solution).
已表明固定化DNA的可及性更关键地取决于固定方法，而非用于固定的支持物类型。当超声处理的变性DNA通过重氮化或通过溴化氰反应偶联到固体葡聚糖G-25和赛利克斯410或大孔琼脂糖凝胶S-500和琼脂糖C1-6B上时，其可及性从100%变化到24%。一般来说，可及性的丧失与在支持物上形成的DNA螺旋的解链温度降低有关。这种相关性对于特定的偶联方法呈现出特征性的变化过程。在变性条件下偶联的DNA，当其仅3%的碱基参与共价连接时，可能会变得完全不可及。对超声处理的大肠杆菌DNA进行的动力学实验表明，通过不同方法固定到固体或大孔支持物上的DNA，其复性或杂交反应的速率常数非常相似。一般来说，异质反应（移动的和固定化的DNA之间）的二级速率常数比类似的均相反应（在溶液中）的二级速率常数小约一个数量级。
