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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis
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Characterization of hairpin-duplex interconversion of DNA using polyacrylamide gel electrophoresis

机译：使用聚丙烯酰胺凝胶电泳表征DNA的发夹-双链互变

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We show how polyacrylamide gel electrophoresis of radiolabeled DNA can be used to measure the hairpin-duplex equilibrium constant for DNA in solution. As an aid to the interpretation of the experiments, the differential equations associated with diffusion, migration and chemical reaction of the DNA forms are solved and intensity patterns generated. Two kinds of experiments were performed on several DNA 12-mers: in the first, electrophoresis time was constant while DNA concentration varied; in the other, concentration was constant while time varied (a `load-and-run' gel). The observed patterns depended on the gel temperature and not the temperature at which the DNA was equilibrated before loading in the well, because reequilibration occurs before the DNA leaves the well to enter the gel proper. During this time, mixing also occurs, changing the concentration and ionic strength of the sample. A method of calculating the true DNA concentration, including the unmeasured concentration added with the radiolabel, is given. When the intensity pattern consists mainly of monomer and dimer peaks, the equilibrium constand K is easily calculated from peak intensities. However, when there is significant intensity between the peaks (which the calculations show results from monomer-dimer interconversion in the gel), K will be inaccurate. An accurate value of K may be determined from a load-and-run gel by extrapolating back to time 0. When the intensity pattern consists of a single broad peak (from rapid monomer-dimer interconversion in the gel), K cannot be calculated without additional information. The rate of interconversion increases with temperature. Estimated rates in the gel are more than an order of magnitude smaller than in bulk solution at the same temperature. Derived values of K for several DNAs are compared with literature values.
机译：我们展示了如何使用放射性标记的DNA的聚丙烯酰胺凝胶电泳来测量溶液中DNA的发夹-双链体平衡常数。为了帮助解释实验，解决了与DNA形式的扩散，迁移和化学反应相关的微分方程，并生成了强度图。在几种DNA 12-聚体上进行了两种实验：首先，电泳时间是恒定的，而DNA浓度是变化的。在另一种情况下，浓度是恒定的，而时间却是变化的（“上样”凝胶）。观察到的模式取决于凝胶温度，而不取决于DNA加载到孔中之前的平衡温度，因为重新平衡发生在DNA离开孔进入合适的凝胶之前。在此期间，还会发生混合，从而改变样品的浓度和离子强度。给出了一种计算真实DNA浓度的方法，包括未测出的放射性标记浓度。当强度模式主要由单体峰和二聚体峰组成时，可以很容易地从峰强度计算出平衡坐标K。但是，当峰之间存在明显的强度时（计算表明凝胶中单体-二聚体相互转化的结果），K将不准确。准确的K值可以通过上载并运行的凝胶通过外推回到时间0来确定。当强度模式由单个宽峰组成（通过凝胶中的快速单体-二聚体相互转化）时，如果没有，则无法计算K附加信息。互变速率随温度而增加。在相同温度下，凝胶中的估计速率比本体溶液中的估计速率小一个数量级以上。将几种DNA的K推导值与文献值进行比较。

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