Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Chuo-ku, Kobe, Japan.
Anal Chem. 2011 Aug 15;83(16):6368-72. doi: 10.1021/ac201312d. Epub 2011 Jul 20.
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.
DNA 芯片将 DNA 寡核苷酸固定在固体板表面上，用于许多诊断应用。为了最大限度地提高检测灵敏度和准确性，控制芯片表面上的 DNA 密度并建立优化密度的便捷方法非常重要。在这里，研究了 DNA 错配结合蛋白 MutS 与芯片表面上的 DNA 底物的结合，该结合可应用于基因组中单核苷酸多态性的高通量分析。我们通过使用混合 DNA 溶液制备了 DNA 芯片，其中 DNA 底物密度的改变很简单。MutS 的结合受到芯片上 DNA 底物量的显著影响，这是由于空间拥挤造成的，并且适当的中等密度使 DNA 底物之间的距离大于蛋白质的大小，从而获得准确的动力学参数。使用混合 DNA 溶液制备的受底物密度控制的 DNA 芯片对于最大限度地提高 MutS 及其可能还有其他大型蛋白质结合的检测能力和动力学分析具有独特的优势。
