Analytical Sciences

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

Ayaka SEICHI,* Nanami KOZUKA,* Yuko KASHIMA,* Miyuki TABATA,** Tatsuro GODA,** Akira MATSUMOTO,** Naoko IWASAWA,* Daniel CITTERIO,* Yuji MIYAHARA,** and Koji SUZUKI*

*Department of Applied Chemistry, Graduate School of Science and Engineering, Keio University, 3-14-1 Hiyoshi, Kohoku, Yokohama, Kanagawa 223-8522, Japan
**Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda Surugadai, Chiyoda, Tokyo 101-0062, Japan

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.

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