2004 Fiscal Year Final Research Report Summary
Manipulation and dissection ofchromosomal DNAbased on micro electro mechanical techniques.
| Project/Area Number |
15011206
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Kyoto University |
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Principal Investigator |
KABATA Hiroyuki Kyoto University, Graduate School of Medicine, Associate professor, 医学研究科, 科学技術振興助教授 (70293884)
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| Co-Investigator(Kenkyū-buntansha) |
KOTERA Hidetoshi Kyoto University, Graduate School of Engineering, Professor, 工学研究科, 教授 (20252471)
WASHIZU Masao the University of Tokyo, Graduate School of Engineering, Professor, 工学系研究科, 教授 (10201162)
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| Project Period (FY) |
2003 – 2004
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| Keywords | micromachining / DNA array / single-molecule handling / chromosome map / electroosmosis / FISH analysis / genomic engineering / nanobiology |
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| Research Abstract |
Extension of chromosomes, from an entangled coiled coil form to an ordered linear fiber form, is a key for high throughput in FISH and karyotypic inspection. We integrated onto a microscope glass surface multiple functions that handle chromosomes as a cat's cradle string. The functions were designed to consist of three connected micromachinings: the arrayer transporting cells to designated positions; the stretcher extracting chromosomes out of the arrayed cells and displaying them as extended fibers; and the analyzer maintaining the chromosome fibers to increase inspection efficiency.
To fix cells along a defined line, we placed a suspension of cells (from a fission yeast or HeLa) on the arrayer and applied a driving force by electroosmosis or traveling wave. Each cell was drifted to a line of curbs (10-micron dimension) fabricated on the arrayer, docked into each curb, and aligned as designed. The arrayed cells were permeabilized and subjected to electroosmotic shear stress generated b
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y the stretcher. Indeed, the chromosomal DNA of each cell was stretched along the stress vector to form a linear fiber. The obtained chromosome fibers reached lengths of >1 mm (>3 Mbp) beyond the field of view, much longer than those reported for conventional air-dry meniscus methods (0.2 mm). This advantage in chromosome extension can be explained by the controlled stress and solution handling, both of which suppress DNA breakage. To retain chromosome fibers, a colonnade of pillars (10-micron dimension) was fabricated on the analyzer. When variable stress vectors were generated, chromosome fibers approached to pillars and adsorbed onto pillar sides electrostatically. Thus, chromosome fibers were stably suspended between pillars a few microns above the surface, ensuring hybridization by probes with the highest accessibility. Demonstration of FISH analyses for c-myc and EGFR genes is underway in the industry-academia collaboration launched from Priority Areas (C) "Genome Science". (299 words) Less
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Research Products
(13 results)
