Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids

• Published in: Analyst (London) 2011-09, Vol.136 (18), p.3635-3642
• Main Authors: KIVLEHAN, Francine, MAVRE, François, TALINI, Luc, LIMOGES, Benoit, MARCHAL, Damien
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Chemistry; DNA - chemistry; DNA - metabolism; DNA Helicases - metabolism; DNA Probes - chemistry; DNA Probes - metabolism; Electrochemical methods; Electrochemical Techniques - methods; Exact sciences and technology; Fluorescent Dyes - chemistry; General, instrumentation; Intercalating Agents - chemistry; Nucleic Acid Amplification Techniques; Oxidation-Reduction; Spectrometric and optical methods; Transition Temperature
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c1an15289k

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.