Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
Anal Chim Acta. 2010 Jan 11;657(2):163-8. doi: 10.1016/j.aca.2009.10.047.
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.
这项工作涉及使用碳糊电极（CPE）的差分脉冲伏安法和悬汞滴电极（HMDE）的交流伏安法研究 SYBR Green I（SG）与天然 DNA 相互作用。在 CPE 上，随着 SG 浓度的增加，1.0 V 处的峰电流强度减小。在 HMDE 上，随着 SG 浓度的增加，-1.2 V 处的 DNA 峰电流强度也减小。这些电化学结果证实了 SG 嵌入 DNA 双螺旋并改变其构象。通过本工作，通过应用交流和差分脉冲伏安技术实现了不同甲基化分析物的区分。通过用 5-甲基胞嘧啶替代 60%的胞嘧啶来合成富含 GC 的 p53 外显子 5 的扩增子（PCR 产物），并用 SYBR Green 和溴化乙锭（EtBr）在差分脉冲伏安法下进行电化学研究。在存在 SG 和 EtBr 的情况下，未甲基化的外显子 5 与甲基化的外显子 5 相比，观察到相当大的峰电流差异。因此，SG 和 EtBr 都可以作为识别不同 DNA 构象的电化学探针。
