Mutation detection by electrocatalysis at DNA-modified electrodes

Detection of mutations and damaged DNA bases is important for the early dia gnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in full y hybridized duplexes, based on charge transport through DNA films. Gold el ectrodes modified with preassembled DNA duplexes are used to monitor the el ectrocatalytic signal of methylene blue, a redox-active DNA intercalator, c oupled to pFe(CN)6](3-). The presence of mismatched or damaged DNA bases su bstantially diminishes the electrocatalytic signal. Because this assay is n ot a measure of differential hybridization, all single-base mismatches, inc luding thermodynamically stable GT and GA mismatches, can be detected witho ut stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this te chnology in a chip-based format. This system provides a sensitive method fo r probing the integrity of DNA sequences and a completely new approach to s ingle-base mismatch detection.