Single-Molecule Detection of Specific Nucleic Acid Sequences in Unamplified Genomic DNA

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Abstract
A new technique is described for the rapid detection of specific nucleic acid sequences in unamplified DNA samples. The method consists of using two nucleic acid probes complementary to different sites on a target DNA sequence. The two probes are each labeled with different fluorescent dyes. When mixed with a sample containing the target DNA, the two probes hybridize to their respective binding sites on the same target DNA molecule. The sample is then analyzed by a laser-based ultrasensitive fluorescence system capable of detecting single fluorescent molecules at two different wavelength channels simultaneously. Since the probes are bound to the same target DNA molecule, their signals appear simultaneously. Thus, coincident detection of both dyes provides the necessary specificity to detect an unamplified, single-copy target DNA molecule in a homogeneous assay. If the target is not present, only uncorrelated events originating from free probes will be observed at either channel. Phage λ DNA in a background of salmon genomic DNA was detected as a two-dye coincident signal at a relative concentration of one λ molecule per salmon genome. In a control sample, cleavage of the λ DNA between the two probe binding sites eliminated the coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applications to genetic analysis are discussed.