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Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

Abstract

Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

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Figure 1: Schematic representation of the double-stranded toehold exchange mechanism.
Figure 2: Discrimination of SNPs by the dsDNA probe.
Figure 3: Concentration of SNP target needed to generate the same χ as a stoichiometric (relative to probe) amount of intended target.
Figure 4: Characterization of the background, temperature, salinity and time robustness of the probe.
Figure 5: Detection of SNPs in E. coli-derived samples.

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Acknowledgements

The authors thank Eric Klavins for insightful discussion and helpful manuscript preparation suggestions. This work was funded by National Institutes of Health Award 1K99EB015331 to D.Y.Z., by National Science Foundation CAREER Award 0954566 to G.S. and by a Defense Advanced Research Projects Agency Young Faculty Award to G.S.

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S.X.C., D.Y.Z. and G.S. conceived the project and designed the experiments. S.X.C. conducted the experiments. S.X.C., D.Y.Z. and G.S. analysed the data and co-wrote the paper.

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Correspondence to David Yu Zhang or Georg Seelig.

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Chen, S., Zhang, D. & Seelig, G. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA. Nature Chem 5, 782–789 (2013). https://doi.org/10.1038/nchem.1713

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