Studies on the Micro-Surgery of Chromosomes

这项研究的目的是开发新的方法和工具，用于染色体的机械操作，将DNA纤维从染色体中拉伸出来，分析染色体的结构，定位DNA链上的特定基因，以及解剖或修改DNA（染色体的显微手术）。所获得的结果总结如下。1）开发了一种微制造装置，其能够在不施加过度流体动力应力的情况下向物体按需供应蛋白酶及其抑制剂。使用该装置，在细胞分裂周期中出现的高等物种的染色体被部分分解，并且使用激光操作方法，已经表明可以从染色体中拉出连续的DNA纤维。研究表明，激光局部加热对染色体的部分分解是有效的。2）提出了一种批量处理酵母细胞总染色体的方法。细胞的细胞骨架使用分子连接物加强以形成包含DNA的笼，通过添加表面活性剂使细胞膜破裂，并应用FISH（荧光原位杂交）方案。然后将细胞放置在固体表面上，部分破坏笼状结构，并使用电渗流，将DNA纤维拉出并拉伸。3）使用电子束沉积（EBD）在玻璃针的尖端上生长微小探针（直径几十nm）。实验表明，可以从固定在疏水表面上的DNA纤维上解剖并拾取目标部分。本研究开发的装置和方法将成为生物纳米技术的有力工具，这将为传统的基于试管的生物化学带来新的“空间分辨率”维度。

The purpose of this research is to develop novel methods and tools for mechanical manipulation of chromosomes, to stretch DNA fibers out of a chromosome, to analyze the structure of a chromosome, to locate a specific gene on the DNA strand, and to dissect or modify DNA (micro surgery of chromosomes). The results obtained are summarized as below.1) A microfabricated device is developed, which enables on-demand supply of protease and its inhibitor to the object without applying excessive hydrodynamic stress. Using the device, a chromosome of higher species which appears in the division cycle of a cell is partially decomposed, and with the use of laser-manipulation methods, it has been shown that a continuous DNA fiber can be pulled out of a chromosome. It is also shown that local heating by laser is effective in partially decomposing a chromosome.2) A method is developed to handle total chromosome of a yeast cell in a batch. The cytoskeleton of the cell is reinforced using molecular linkers to form a cage in which DNA is contained, cell membrane ruptured by adding surfactants, and FISH (Fluorescent in situ Hybridization) protocol is applied. Then the cell is placed on a solid surface, the cage structure partially destroyed, and using electroosmotic flow, DNA fiber is pulled out and stretched.3) Electron Beam Deposition (EBD) is used to grow a tiny probe (several ten nm in diameter) on the tip of a glass needle. It is experimentally shown that aimed portion can be dissected and picked up from DNA fibers immobilized on a hydrophobic surface. The devices and methods developed in this research will be a powerful tools for bio-nanotechnology, which will bring about a new dimension of "spatial resolution" to conventional test-tube based biochemistry.