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Ultrasensitive coincidence fluorescence detection of single DNA molecules.

Authors
  • Li, Haitao
  • Ying, Liming
  • Green, Jeremy J
  • Balasubramanian, Shankar
  • Klenerman, David
Type
Published Article
Journal
Analytical chemistry
Publication Date
Apr 01, 2003
Volume
75
Issue
7
Pages
1664–1670
Identifiers
PMID: 12705600
Source
Medline
License
Unknown

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.

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