Hybridization of mismatched or partially matched DNA at surfaces
Hybridization of mismatched or partially matched DNA at surfaces
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DOI：
10.1021/ja0279996
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发表时间：
2002-12-11
影响因子：
15
通讯作者：
Georgiadis, RM
中科院分区：
文献类型：
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作者：
Peterson, AW;Wolf, LK;Georgiadis, RM
我们研究探针密度如何影响包含不匹配序列的未标记目标寡核苷酸或进入固定探针上不同结合位置的目标的杂交。我们发现强探针密度效应不仅影响杂交效率，而且影响捕获动力学。探针表面被反复使用，并解决了样品间表面异质性和非特异性吸附变化的潜在大贡献。利用原位表面等离子体共振（SPR）光谱获得的动力学、平衡和温度依赖研究结果表明，表面固定化DNA的杂交与研究得很好的溶液相反应有很大不同。表面杂交很大程度上取决于靶序列和探针密度。许多数据可以用高探针密度时空间拥挤的存在来解释；然而，即使在研究的最低密度下，使用标准杂交模型也无法理解错配序列的行为。除了不寻常的捕获动力学观察到的不匹配的目标，我们发现，结合等温线可以拟合只有当一个异质模型被使用。对于不匹配的靶标，Sips模型充分描述了探针-靶标结合等温线；对于完全匹配的目标，可以使用Langmuir模型。
We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.