Real-time monitoring and detection of primer generation-rolling circle amplification of DNA using an ethidium ion-selective electrode

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 generationrolling 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.