Real-time Monitoring and Detection of Primer Generation-Rolling Circle Amplification of DNA Using an Ethidium Ion-selective Electrode

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  • SEICHI Ayaka
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University
  • KOZUKA Nanami
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University
  • KASHIMA Yuko
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University
  • TABATA Miyuki
    Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
  • GODA Tatsuro
    Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
  • MATSUMOTO Akira
    Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
  • IWASAWA Naoko
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University
  • CITTERIO Daniel
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University
  • MIYAHARA Yuji
    Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
  • SUZUKI Koji
    Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University

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Abstract

An electrochemical detection system for an isothermal DNA amplification method using an ion-selective electrode (ISE) was developed as a low-cost, simple and real-time monitoring system. The system is based on potentiometry using an ethidium ion (Et+) selective electrode that relies on monitoring DNA amplification by measuring potential changes in the reaction solution containing ethidium bromide (EtBr) as an intercalator to DNA. With progressing primer generation-rolling circle amplification (PG-RCA) under isothermal condition at 37°C, EtBr is bound to the newly formed DNA, resulting in a lowered free EtBr concentration in the sample solution. In this case, the Et+ ISE potential allows real-time monitoring of the PG-RCA reaction in the range of 10 nM – 1 μM initial target DNA.

Journal

  • Analytical Sciences

    Analytical Sciences 32 (5), 505-510, 2016

    The Japan Society for Analytical Chemistry

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