KINETICS OF RENATURATION OF DNA
KINETICS OF RENATURATION OF DNA
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DOI：
10.1016/0022-2836(68)90414-2
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发表时间：
1968-01-01
影响因子：
5.6
通讯作者：
DAVIDSON, N
中科院分区：
文献类型：
--
作者：
WETMUR, JG;DAVIDSON, N
完全变性的DNA的复性速率在动力学上是二级反应。反应速率随着温度降低至Tm（50%变性温度）以下而增加，在低于Tm 15至30 ℃时达到一个宽平的最大值，然后随着温度的进一步降低而降低。设N为DNA的复杂性或给定DNA的每个病毒或细胞的非重复序列中的碱基对数量，L为变性DNA制备物的每条单链的平均核苷酸数量。然后，所有DNA的二级复性速率常数近似由k2 =3 × 105 L 0.5/N 1给出。在（Tm-25）[degree]C和[Na+] =1.0 mole l.-1 sec-1 [其中L是长度，N是复杂性]下1在水溶液中。反应速率随着DNA中GC含量的增加而略有增加。当粘度通过添加对Tm具有小的影响（蔗糖、甘油、乙二醇）或大的影响（NaClO 4）的组分而改变时，在最大温度（Tm[长划线]25）[℃]下的反应速率与溶剂粘度成反比。有人提出，该反应的机制涉及的2条链上的短，同源位点的加入，然后由一个快速，可逆的拉链反应与正向速率常数kf。该模型的计算机分析解释了温度和GC依赖性。为了解释粘度的依赖性，建议KF是成反比的粘度，也就是说，拉链反应是流体动力学限制。任何简单的理论都可以预测k2 [长破折号]L/N;观察到的L0.5长度依赖性归因于排除的体积或空间位阻效应，即2个互补变性DNA螺旋的限制性互穿。
The rate of renaturature of fully denatured DNA is kinetically a 2nd-order reaction. The reaction rate increases as the temperature decreases below Tm [temperature for 50% denaturation], reaching a broad flat maximum from 15 to 30[degree]C below Tm and then decreases with a further decrease in temperature. Let N be the complexity of the DNA or the number of base-pairs in nonrepeating sequences per virus or cell for the given DNA, and L the average number of nucleotides per single strand of the denatured DNA preparation. Then, the 2nd-order renaturation rate constants for all DNA''s are given approximately by k2 =3 x 105 L0.5/N l. mole-1 sec-1 [where L is length and N is complexity] at (Tm-25)[degree]C and at [Na+] =1.0 mole l.-1 in aqueous solution. The reaction rate increases slightly with the GC content of the DNA. The reaction rate at the temperature maximum (Tm[long dash]25)[degree]C is inversely proportional to solvent viscosity, when the viscosity is changed by the addition of components which either have a small (sucrose, glycerol, ethylene glycol) or a large (NaClO4) effect on Tm. It is proposed that the mechanism of the reaction involves the joining of short, homologous sites on the 2 strands followed by a fast, reversible zippering reaction with forward rate constant kf. A computer analysis for this model explains the temperature and the GC dependence. To explain the viscosity dependence it is proposed that kf is inversely proportional to viscosity; that is, the zippering reaction is hydrodynamically limited. Any simple theory predicts k2 [long dash]L/N; the observed L0.5 length dependence is attributed to an excluded volume or steric hindrance effect, that is, to restricted interpenetration of the 2 complementary denatured DNA coils.