Studies on the Micro-Surgery of Chromosomes

点击翻译按钮获取中文摘要

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.