Electroanalytical study of SYBR Green I and ethidium bromide intercalation in methylated and unmethylated amplicons.

This work involves the electrochemical study of the interaction of SYBR Green I (SG) with native DNA using differential pulse voltammetry at a carbon paste electrode (CPE) and alternating current voltammetry at a hanging mercury drop electrode (HMDE). At the CPE the peak current intensity at 1.0 V decreased by increasing the concentration of SG. At the HMDE, a decrease in the current intensity of the DNA peak at -1.2 V was also observed by increasing the concentration of SG. These results electrochemically confirmed that SG intercalates within the DNA double helix and changes its conformation. Through the present work the differentiation of differently methylated analytes was achieved by application of alternative current and differential pulse voltammetric techniques. Amplicons (PCR products) corresponding to the GC-rich p53 exon 5 containing cytosine and its methylated analogue, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, were amplified and investigated electrochemically in the presence of SG and ethidium bromide (EtBr) by differential pulse voltammetry. Considerable peak current differences were observed in the presence of SG and EtBr for unmethylated exon 5 vs. methylated. Therefore, both SG and EtBr could serve as electrochemical probes for identifying different DNA conformations.