Kyohei Terao

Extending chromosomal DNA in microstructures using electroosmotic flow

The extension of chromosomal DNA is a key technique in high-resolution gene location such as extended fibre fluorescent in situ hybridization. In this paper, we propose a new micro-device designed for on-chip integration of three functions, (1) positioning of cells, (2) extending chromosomal DNA from the cells, and (3) anchoring the extended DNA fibres. The device has a flow chamber equipped with (a) multi-phase electrodes to create electroosmotic flow (EOF) of variable direction, (b) a set of micro-fabricated pocket-like structures for cell positioning, and (c) a micro-pillar array whose surface is chemically modified to anchor DNA fibres. First, unidirectional EOF is induced, by which cells are carried into the micro-pockets and positioned, one cell at each pocket. After rupturing the cells, chromosomal DNA from each cell is hydrodynamically extended by EOF. When the EOF direction is rotated through 90°, the fibres are made to contact with the pillars to be anchored. Because the velocity of the EOF is controllable, the breakage of DNA during the process can be minimized. Thus immobilized DNA fibres are suspended a few microns above the surface, allowing free access of probe molecules.

Complete Extension of Chromosomal DNA and Its Manipulation Using Optically-Driven Micro-Structures

This paper presents a novel method for extension and manipulation of chromosomal DNA using electroosmotic flow (EOF) and optically-driven micro-structures, which is intended for the use in nanotechnology-based genomic studies. Chromosomal DNA fibers are first hydrodynamically extended to the full length between micro-pillars on a substrate by EOF. In order to manipulate single chromosomal DNA fibers, we developed micro-hook and micro-bobbin, which were fabricated with SU-8 photo resist and grasped by focused laser beams. A targeted position of an extended DNA fiber was picked up with the micro-hook and translocated by moving the hook. To manipulate a whole DNA fiber, an extended fiber was wound to be compact using micro-bobbins. These handling techniques realize the observation of single chromosomal DNA molecules with high spatial resolution

Manipulation of yeast chromosomal DNA using optically driven microstructures

This paper presents a novel method for manipulating single chromosomal DNA, which is intended for the use in highresolution genomic studies. Such operations as translocation, winding and unwinding of single DNA fiber are achieved using optically-driven micro-fabricated structures, including micro-hooks and micro-bobbins for picking-up and winding DNA, with a typical dimension of several μm. The geometry of the laser-manipulated micro-structures is designed in such a way that a spontaneous orientation occurs with its major axis parallel to the laser beam and accepts a DNA fiber. While monitoring under a fluorescence microscope, yeast chromosomal DNA is first extended to the full length by electroosmotic flow. Then the micro-hooks are dispensed in the solution, and a DNA fiber is picked up with the microhook which is driven by a focused laser beam, to separate the targeted DNA from the others. The winding is achieved with a pair of micro-bobbins. The laser is split into two, the first beam being fixed, and the second movable circularly around the first. When the bobbins are made into contact with DNA and revolving motion started, the fiber is wound and suspended between them. The unwinding can be achieved just by reversing the revolving motion.