Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

An immobilization-free electrochemical method is reported for real-time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully-complementary or single-base-mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (]TO). When a ferrocene labeled PNA (Fc-PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the FcPNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc-PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc-PNA/DNA hybrid can be obtained by measuring the Fe signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.