SINGLE-MOLECULE DETECTION OF SPECIFIC NUCLEIC-ACID SEQUENCES IN UNAMPLIFIED GENOMIC DNA

A new technique is described for the rapid detection of specific nucle ic acid sequences in unamplified DNA samples. The method consists of u sing two nucleic acid probes complementary to different sites on a tar get DNA sequence. The two probes are each labeled with different fluor escent dyes. When mixed with a sample containing the target DNA, the t wo probes hybridize to their respective binding sites on the same targ et DNA molecule. The sample is then analyzed by a laser-based ultrasen sitive fluorescence system capable of detecting single fluorescent mol ecules 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 u ncorrelated events originating from free probes will be observed at ei ther channel. Phage lambda DNA in a background of salmon genomic DNA w as detected as a two-dye coincident signal at a relative concentration of one lambda molecule per salmon genome. In a control sample, cleava ge of the lambda DNA between the two probe binding sites eliminated th e coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applic ations to genetic analysis are discussed.