Journal
NATURE CHEMISTRY
Volume 5, Issue 9, Pages 782-789Publisher
NATURE RESEARCH
DOI: 10.1038/NCHEM.1713
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Funding
- National Institutes of Health Award [1K99EB015331]
- National Science Foundation CAREER Award [0954566]
- Defense Advanced Research Projects Agency Young Faculty Award
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [0954566] Funding Source: National Science Foundation
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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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