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Mutation detection by electrocatalysis at DNA-modified electrodes

Nature Biotechnology volume 18, pages 1096–1100 (2000)Cite this article

Abstract

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.

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Figure 1: Schematic representation of electrocatalytic reduction of [Fe(CN)6]3− by LB+ at a DNA-modified electrode.
Figure 2: Chronocoulometry at −350 mV of 2.0 mM [Fe(CN)6]3− plus 0.5 μM MB+ (pH 7) at a gold electrode modified with the thiol-terminated sequence SH-5′-AGTACAGTCATCGCG hybridized to a fully base-paired complement (TA) and complements that introduce single-base mismatches.
Figure 3: Chronocoulometry at −350 mV of 2.0 mM [Fe(CN)6]3− plus 0.5 μM MB+ (pH 7) at a gold electrode modified with the thiol-terminated sequence SH-5′-ATGGGCCTCCGGTTC hybridized to a fully base-paired wild-type complement (WT, red) and complements that feature mutations at the boldface C and the underlined C.
Figure 4: Chronocoulometry at −350 mV of 2.0 mM [Fe(CN)6]3− plus 0.5 μM MB+ (pH 7) at a gold electrode modified with the thiol-terminated sequence SH-5′-AGTACAGTCATCGCG hybridized to a fully base-paired complement (TA) and complements that introduce single-base lesions.
Figure 5: Plot of total amount of charge accumulated after 5 s of chronocoulometry at –350 mV of 2.0 mM [Fe(CN)6]3− plus 0.5 μM MB+ (pH 7) at gold electrodes ranging in diameter from 30 μm to 500 μm modified with the thiol-terminated sequence SH-5′-AGTACAGTCATCGCG hybridized to a fully base-paired complement.
Figure 6: Chronocoulometry at −350 mV of 2.0 mM [Fe(CN)6]3− plus 0.5 μM MB+ (pH 7) at a 500 μm gold electrode modified with the thiol-terminated sequence SH-5′-AGTACAGTCATCGCG hybridized either (1) to a fully base-paired complement (TA); (2) to a complement with a single base pair mismatch (CA); or the single stranded monolayer formed by denaturation (ss).

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Acknowledgements

We are grateful to the NIH for financial support (GM61077). We also thank M.J. Fitzsimmons and J.L. Lamb in Device Research at JPL for assistance in the fabrication of the DNA chips.

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Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, CA

    Elizabeth M. Boon, Donato M. Ceres, Thomas G. Drummond & Jacqueline K. Barton

  2. Department of Chemistry, Occidental College, Los Angeles, 90041, CA

    Michael G. Hill

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  1. Elizabeth M. Boon
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  2. Donato M. Ceres
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  4. Michael G. Hill
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  5. Jacqueline K. Barton
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Correspondence to Michael G. Hill or Jacqueline K. Barton.

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Boon, E., Ceres, D., Drummond, T. et al. Mutation detection by electrocatalysis at DNA-modified electrodes. Nat Biotechnol 18, 1096–1100 (2000). https://doi.org/10.1038/80301

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