Theoretical analysis of the footprinting experiment.
Theoretical analysis of the footprinting experiment.
复制标题
足迹实验的理论分析。
DOI：
10.1080/07391102.1985.10507613
复制
发表时间：
1985
影响因子：
4.4
通讯作者：
Dabrowiak,JC
中科院分区：
文献类型：
--
作者：
Goodisman,J;Dabrowiak,JC
在足迹实验中，末端放射性标记的DNA限制片段在配体存在和不存在的情况下被内切酶消化，这改变了内切酶在配体-DNA接触位点的切割速率。这些位点的位置和配体结合的强度，然后从消化产生的不同寡核苷酸的测量浓度中推断出来。我们根据耦合动力学方程来分析实验，该方程考虑了内切酶对配体存在和不存在的位点的切割速率，以及配体与位点的结合和解离速率。只要配体浓度基本保持恒定（例如，如果消化过程足够早地终止，以确保所有片段都是由内切酶的单次切割产生的），寡核苷酸浓度仅反映配体结合平衡常数（速率常数之比）和配体存在和不存在时的切割速率。我们还展示了如何使用测量的寡核苷酸浓度（例如，从自射线图）来推断聚合物上不同位点的配体平衡结合常数。
In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.