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Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3-. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without 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 technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.

Supporting InformationTable S-1. Tabulated electrochemical data for the series of tether lengths investigated. Figure S-1. Variation of cathodic and anodic peak potentials as a function of scan rate (in V/s) for the series of tethers investigated. Black squares: n = 4; red circles: n = 5; blue up triangles: n = 6; dark yellow down triangles: n = 7; purple diamonds: n = 8; green left triangles: n = 9. Laviron formalism was used to generate fit lines for each data set using an α value of 0.6. Background-subtracted cyclic voltammograms were generated by numerically interpolating the charging currents to points just before and after the onset of the respective cathodic and anodic Faradaic responses. The resulting backgrounds were then subtracted from the raw data to yield traces like those shown in Figure 2 of the text. We note that the "trumpet plots" shown above are based on uncorrected voltammograms; use of background-subtracted data instead gave peak splittings (and resulting k s values) within experimental error of those shown. Figure S-2. Representative cyclic voltammograms for two DM-DNA films (n = 5 top, and n = 9, bottom) recorded in 5mM phosphate, 50mM NaCl, pH 7.5 at a series of scan rates. As the scan rate increases, the peak potentials shift slowly for the shorter tether but much more quickly for the longer tether due to the distance-dependence of charge transfer kinetics across the σ-bonded tether.

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