Studies of elementary techniques for developing on-chip devices for single genomic DNA manipulation and analysis

研究开发用于单基因组 DNA 操作和分析的片上设备的基本技术

• 批准号: 17310081
• 负责人: OANA Hidehiro
• 金额: $ 9.7万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17310081/
• 关键词: DNA; single molecule analysis; micro manipulation; optical tweezers; micro fluidic devidce; 単分子操作; ゲノム; マイクロ液体デバイス; 高次構造

Through this research, we developed a new methodology for the observation and the artificial control of higher-order structures of genomic DNA molecules, and the method enables reversible folding and unfolding of over 250 μm-long DNA with a high reproducibility.In eukaryotic cells, DNA with the aid of cationic proteins such as histone are folded to form a highly ordered chromosome structures, and the gene expression is influenced by how tightly it is fold around a particular gene, which is controlled by such a process as histone methylation or acetylation. However, the mechanism and the dynamics of DNA folding, such as the speed of folding on a DNA strand, or how much cooperativity (the folding speed is enhanced once folding starts) influences the folding of very long DNA strand, is not well understood.Such a study requires a method to stretch DNA strands, hold it in a solution, which can be quickly replaced the solutions having different cationic or salt concentrations. For this purpose, we have developed a microfluidic device. The device which we developed is fabricated by a standard soft lithography with PDMS. The main channel of the device has micro-pillars to hook and stretch DNA by hydrodynamic force. There are inlets on the upstream side of the pillars, from which yeast chromosomal DNA, 1 mM spermidine (DNA condensing polycationic agent), and 500 mM NaCl (de-condensing salt) are fed. Each solution contains 1 μM YO-PRO-1 for fluorescent visualization of DNA.The method developed here not only contributes to the basic biochemical researches, but may also open a way for the DNA handling towards single-molecule sequencing, or control of cellular activity through artificially induced higher-order changes.

通过这项研究，我们发展了一种新的方法来观察和人工控制基因组DNA分子的高级结构，该方法能够以高重复性可逆地折叠和展开超过250 μ m长的DNA。在真核细胞中，DNA在阳离子蛋白如组蛋白的帮助下折叠形成高度有序的染色体结构，而基因的表达受其围绕特定基因的紧密程度的影响，这是由组蛋白甲基化或乙酰化等过程控制的。然而，DNA折叠的机制和动力学，例如DNA链上的折叠速度，或者协同性（一旦折叠开始，折叠速度被提高）对非常长的DNA链的折叠有多大影响，还没有很好地理解。这样的研究需要一种方法来拉伸DNA链，将其保持在溶液中，该溶液可以快速地替换为具有不同阳离子或盐浓度的溶液。为此，我们开发了一种微流控装置。我们所开发的器件是由一个标准的软光刻与PDMS。该装置的主通道具有微柱，以通过流体动力钩住和拉伸DNA。在柱的上游侧上有入口，从该入口进料酵母染色体DNA、1 mM亚精胺（DNA缩合聚阳离子剂）和500 mM NaCl（解缩合盐）。每种溶液中含有1 μM YO-PRO-1，可用于DNA的荧光显色。该方法不仅有助于基础生物化学研究，而且可能为DNA处理向单分子测序或通过人工诱导的高阶变化控制细胞活性开辟一条道路。

项目成果

「ゲノムDNAの単分子操作」バイオプロセスを利用した有用物質生産技術ハンドブック

《基因组DNA的单分子操作》利用生物过程的有用物质生产技术手册

• 发表时间: 2007
• 作者: 寺尾京平;小穴英廣;鷲津正夫(分担執筆)
• 通讯作者: 鷲津正夫(分担執筆)

Complete extension of chromosomal DNA and its manipulation using optically-driven micro-structures

染色体 DNA 的完全延伸及其使用光学驱动微结构的操纵

• 发表时间: 2006
• 期刊: 2006 IEEE International Symposium on Micro-Nano Mechatronics and Human Science 2
• 作者: K. Terao;H. Kabata;H. Oana;M. Washizu
• 通讯作者: M. Washizu

光ピンセット用紐状物質捕捉部材

光镊用线状物质捕捉部件

• 发表时间: 2005

COMPLETE EXTENSION OF CHROMOSOMAL DNA AND ITS MANIPULATION USING OPTICALLY-DRIVEN MICRO-FABRICATED HOOKS

染色体 DNA 的完全延伸及其使用光学驱动微制造钩的操作

• 发表时间: 2005
• 期刊: Proc. 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences (Micro TAS 2005) 1
• 作者: K. Terao;H. Kabata;H. Oana and M. Washizu
• 通讯作者: H. Oana and M. Washizu

光駆動微小構造体を用いた染色体DNAの液中1分子操作

利用光驱动微结构对液体中染色体 DNA 进行单分子操控

• 发表时间: 2006
• 作者: 寺尾京平;加畑博幸;小穴英廣;鷲津正夫
• 通讯作者: 鷲津正夫