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Studies on the Micro-Surgery of Chromosomes

染色体显微外科研究

基本信息

批准号：
11450103
负责人：
WASHIZU Masao
金额：
$ 8.58万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

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.

本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，从染色体中拉伸DNA纤维，分析染色体的结构，定位DNA链上的特定基因，以及解剖或修饰DNA（染色体的微外科手术）。所得结果总结如下。1)开发了一种微加工装置，可以在不施加过大流体动力应力的情况下按需供应蛋白酶及其抑制剂。利用该装置，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，已经证明可以从染色体中拔出连续的DNA纤维。激光局部加热对染色体的部分分解是有效的。研究了一种批量处理酵母细胞总染色体的方法。使用分子连接物加强细胞骨架，形成一个包含DNA的笼子，细胞膜通过添加表面活性剂破裂，并应用FISH（荧光原位杂交）协议。然后将细胞置于固体表面，部分破坏笼状结构，利用电渗透流，将DNA纤维拉出并拉伸。电子束沉积（EBD）用于在玻璃针的尖端生长一个微小的探针（直径几十纳米）。实验表明，目标部分可以从固定在疏水表面的DNA纤维中分离和提取。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，为传统试管生物化学带来一个新的“空间分辨率”。