title: Ultrasensitive coincidence fluorescence detection of single DNA molecules.
abstract: We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
authors.forename: Haitao
authors.initials: H
authors.lastname: Li
authors.forename: Liming
authors.initials: L
authors.lastname: Ying
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Green
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D005456:Fluorescent Dyes; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods*; D012680:Sensitivity and Specificity; D012996:Solutions / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D012996:Solutions; D004247:DNA