A random-walk model for retardation of interacting species during gel electrophoresis: Implications for gel-shift assays
A random-walk model for retardation of interacting species during gel electrophoresis: Implications for gel-shift assays
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DOI：
10.1016/s0006-3495(97)78162-1
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发表时间：
1997-09-01
影响因子：
3.4
通讯作者：
Johnston, BH
中科院分区：
文献类型：
--
作者：
Belotserkovskii, BP;Johnston, BH
我们最近表明，当较快迁移的单链超过包含适当序列双链体的较慢迁移带时，分子间 DNA 三链体可以在凝胶电泳过程中形成。我们提出了一个模型来解释当三联体的寿命比电泳时间短得多时所产生的三联体与双链体带的明显共校准。该模型预测，如果复合物中迁移较快的组分被标记，迁移较慢的组分过量，并且复合物本身比任一组分迁移得更慢，则可以通过凝胶迁移测定检测短寿命的复合物。在这种情况下，标记的组分在从复合物解离后，超越游离的、未标记的第二组分的较慢迁移带，并且可以被未标记的组分捕获并再次被延迟；新形成的复合物解离后，重复该循环。如果条带中未标记组分的浓度大于某个临界值 (c(cr))，则在凝胶电泳的整个时间内，大部分标记组分都会被捕获在该条带中，从而有效地与迁移较慢的未标记组分共迁移。我们将这种共迁移机制称为“循环捕获和解离”(CCD)。在这里，我们对 CCD 共移模型进行了定量分析，预测 CCD 共移不仅可以用于检测相对较短寿命的复合物，还可以用于估计复合物形成的特异性。
We recently showed that intermolecular DNA triplexes can form during gel electrophoresis when a faster migrating single strand overtakes a slower migrating band containing a duplex of appropriate sequence. We proposed a model to account for the resulting apparent comigration of triplexes with the duplex band when the lifetime of the tripler is much shorter than the time of electrophoresis. The model predicts that short-lived complexes can be detected by a gel-shift assay if the faster migrating component of the complex is labeled, a slower migrating component is in excess, and the complex itself migrates more slowly than either of the components. In this case the labeled component, after dissociation from the complex, overtakes a slower migrating band of the free, unlabeled second component and can be captured by the unlabeled component and again retarded; after dissociation of the newly formed complex the cycle is repeated. If the concentration of unlabeled component in the band is larger than some critical value (c(cr)), most of the labeled component becomes trapped in this band during the entire time of gel electrophoresis, thus effectively comigrating with the slower migrating unlabeled component. We call this mechanism of comigration ''cyclic capture and dissociation'' (CCD). Here we present a quantitative analysis of the model of CCD comigration which predicts that CCD comigration can be used not only for the detection of relatively short-lived complexes, but also for estimation of the specificity of complex formation.