Matsuura Shun-ichi, Kurita Hirofumi, Nakano Michihiko, Komatsu Jun, Takashima Kazunori, Katsura Shinji, Mizuno Akira
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Aichi, 441-8580, Japan.
J Biomol Struct Dyn. 2002 Dec;20(3):429-36. doi: 10.1080/07391102.2002.10506861.
We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. lambda DNA molecules were ligated with 3'-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified lambda DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified lambda DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.
我们展示了一种将DNA固定在疏水玻璃表面的有效方法。与传统技术相比，这种新的DNA固定技术极其简单，传统技术需要在DNA和均经过化学修饰的底物表面之间使用异双功能交联剂。在第一个步骤中，用二氯二甲基硅烷处理盖玻片，形成疏水表面。λDNA分子在一个粘性末端与3'-末端二硫化物修饰的14聚体寡核苷酸连接。二硫化物还原为巯基（硫醇）基团后，所得的硫醇修饰的λDNA分子在硅烷化盖玻片上反应。荧光观察表明，硫醇修饰的λDNA分子在一个末端特异性地锚定在疏水表面，尽管DNA分子的非特异性结合受到抑制。观察到，当施加直流电场时，一端附着的DNA分子牢固地结合在表面并在一个方向上可逆地伸展。
