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

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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.