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Detection of single-base DNA mutations by enzyme-amplified electronic transduction

Nature Biotechnology volume 19, pages 253–257 (2001)Cite this article

Abstract

Here we describe a method for the sensitive detection of a single-base mutation in DNA. We assembled a primer thiolated oligonucleotide, complementary to the target DNA as far as one base before the mutation site, on an electrode or a gold–quartz piezoelectric crystal. After hybridizing the target DNA, normal or mutant, with the sensing oligonucleotide, the resulting assembly is reacted with the biotinylated nucleotide, complementary to the mutation site, in the presence of polymerase. The labeled nucleotide is coupled only to the double-stranded assembly that includes the mutant site. Subsequent binding of avidin–alkaline phosphatase to the assembly, and the biocatalyzed precipitation of an insoluble product on the transducer, provides a means to confirm and amplify detection of the mutant. Faradaic impedance spectroscopy and microgravimetric quartz-crystal microbalance analyses were employed for electronic detection of single-base mutants. The lower limit of sensitivity for the detection of the mutant DNA is 1 × 10−14 mol/ml. We applied the method for the analysis of polymorphic blood samples that include the Tay–Sachs genetic disorder. The sensitivity of the method enables the quantitative analysis of the mutant with no PCR pre-amplification.

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Figure 1
Figure 2: Amplified detection of the DNA mutant (2) by the biocatalyzed precipitation of (5) using Faradaic impedance spectroscopy.
Figure 3: Calibration curve corresponding to changes in the electron transfer resistances of the electrode as a result of the precipitation of (5) upon detection of different concentrations of (1).
Figure 4: (A) Faradaic impedance spectra (Zim vs. Zre) corresponding to (a) the (3)-functionalized electrode itself and (b) after treatment with 3 × 10−9 mol/ml of normal oligonucleotide (2).
Figure 5: Time-dependent frequency changes upon the association of avidin–alkaline phosphatase and the biocatalyzed precipitation of (5) in the presence of the oligonucleotide/DNA assemblies:

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Acknowledgements

Parts of the research are supported by the Max Planck Research Award for International Cooperation and by the Israel Ministry of Science as an Infrastructure Project in Biomicroelectronics.

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  1. Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel

    Fernando Patolsky, Amir Lichtenstein & Itamar Willner

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  1. Fernando Patolsky
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Correspondence to Itamar Willner.

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Patolsky, F., Lichtenstein, A. & Willner, I. Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat Biotechnol 19, 253–257 (2001). https://doi.org/10.1038/85704

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