Microfluidics-based in situ Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

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  • KURODA Arisa
    Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University
  • ISHIGAKI Yuri
    Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University
  • NILSSON Mats
    Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University
  • SATO Kiichi
    Division of Molecular Science, School of Science and Technology, Gunma University
  • SATO Kae
    Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University

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タイトル別名
  • Microfluidics-based <i>in situ</i> Padlock/Rolling Circle Amplification System for Counting Single DNA Molecules in a Cell

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説明

In situ padlock/rolling circle amplification (RCA) is a method used to amplify, visualize, and quantify target DNA molecules in cells. However, the multiple reaction steps involved make this technique costly and cumbersome. We developed a novel, simplified, automated microfluidic system for RCA, and demonstrated its effectiveness by counting amplified mitochondrial DNA fragments in HeLa cells. After optimizing the volume of the reaction solutions and washing buffer composition, the product yield was equal to that obtained by the conventional manual method. The required volume of reagents was reduced to 10 μL, which is less than half the volume used in the conventional method. To the best of our knowledge, this is the first report of an automated microfluidic method for in situ padlock/RCA, which can be useful for making highly efficient pathological diagnoses.

収録刊行物

  • Analytical Sciences

    Analytical Sciences 30 (12), 1107-1112, 2014

    社団法人 日本分析化学会

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