Electrochemical DNA base pairs quantification and endonuclease cleavage detection

A new electrochemical approach for an accurate quantification of DNA base pairs in genomic human DNA amplified by polymerase chain reaction (PCR) is described. The method is based on the immobilization of the sample (a thiolated DNA fragment) on the surface of a screen-printed gold electrode through the -SH group at the 5'-end and the subsequent intercalation of a ruthenium pentaamin complex as a redox indicator. The determination of the base pair number in the sequence is achieved by measuring the changes in the electroactivity of the ruthenium complex using Differential Pulse Voltammetry. Calibration curves correlating current intensity with the base pair number allow determining the size of DNA samples, even when very large (over 100 base pairs) sequences are assayed. The method has been successfully applied to detect the DNA cleavage by a site-specific restriction enzyme. The electrochemical approach developed offers the advantage of ease of performance in comparison to other previously described approaches, which are time-consuming and require sophisticated and expensive instrumentation. (C) 2011 Elsevier B.V. All rights reserved.