title: 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.
authors: Department of Electrical Engineering, University of Washington, Seattle, Washington 98195, USA.
authors: Sherry Xi
authors: SX
authors: Chen
authors: David Yu
authors: DY
authors: Zhang
authors: Georg
authors: G
authors: Seelig
mesh_terms: D020137:Base Pair Mismatch; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000737:chemistry* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D054712:Salinity; D013696:Temperature
chemical_list: D015342:DNA Probes; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C540081:rpoB protein, E coli; D004247:DNA; D012321:DNA-Directed RNA Polymerases
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