Measurements of the binding of a large protein using a substrate density-controlled DNA chip
Measurements of the binding of a large protein using a substrate density-controlled DNA chip
复制标题
使用底物密度控制 DNA 芯片测量大蛋白质的结合
DOI：
10.1021/ac201312d
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
N.Sugimoto
中科院分区：
文献类型：
--
作者：
S.Nakano;T.Kanzaki;M.Nakano;D.Miyoshi;N.Sugimoto
将DNA寡核苷酸固定在固体板表面上的DNA芯片用于许多诊断应用。为了最大化检测灵敏度和准确度，重要的是控制芯片表面上的DNA密度并建立用于优化密度的方便方法。在此，研究了DNA错配结合蛋白MutS与芯片上的DNA底物的结合，其可应用于基因组中的高通量单核苷酸多态性分析。我们制备了DNA芯片，其中通过使用混合DNA溶液简单地改变DNA底物密度。MutS的结合受到芯片上的DNA底物的量的显著影响，作为空间拥挤的结果，并且使DNA底物之间的距离大于蛋白质的大小的中等密度适于获得准确的动力学参数。使用混合DNA溶液制备的底物密度控制的DNA芯片具有显著的优点，用于最大化MutS和可能还有其他大蛋白的结合的检测能力和动力学分析。
The DNA chip that immobilizes DNA oligonucleotides on a solid plate surface is used for many diagnostic applications. For maximizing the detection sensitivity and accuracy, it is important to control the DNA density on a chip surface and establish a convenient method for optimizing the density. Here, the binding of DNA mismatch-binding protein MutS to the DNA substrate on the chip was investigated, which can be applied for high-throughput single-nucleotide polymorphism analysis in a genome. We prepared the DNA chips where the DNA substrate density was changed simply by using a mixed DNA solution. The binding of MutS was significantly influenced by the amount of DNA substrate on the chip as a consequence of steric crowding, and the moderate density that gave the distance between the DNA substrates greater than the size of the protein was appropriate to obtain accurate kinetic parameters. The substrate density-controlled DNA chip prepared using the mixed DNA solution has distinctive advantages for maximizing the detection capability and kinetic analysis of the binding of MutS and probably also other large proteins.