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Proceedings Paper

Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection
Author(s): Peter M. Goodwin; Rhiannon L. Nolan; Hong Cai
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Paper Abstract

Single-molecule fluorescence techniques are used to detect a specific nucleic acid sequence in a mixture of unrelated sequences. Co-hybridization of a pair oligonucleotide hybridization probes, each labeled with a spectrally-distinct fluorophore and complementary to a specific sub-sequence of the target nucleic acid, forms a fluorescent adduct containing both fluorophores. The presence of the specific sequence is signaled by the simultaneous detection of both fluorophore labels on a single target fragment. We demonstrate quantitative detection of target nucleic acid sequences at fragment concentrations as low as 100 fM with a simple instrument that uses low-power, continuous-wave laser excitation. Furthermore, we show that a cross-correlation analysis of the arrival times of individual single-molecule fluorescence photon bursts detected in spectrally separate channels permits quantitative detection of the dual-color labeled species at concentrations approximately 1000x lower than can be quantitatively detected using the photon cross-correlation between the two detection channels. We also demonstrate that a pair of quencher-labeled oligonucleotides each complementary to the fluorescent hybridization probes can be used to reduce unbound probe fluorescence, substantially improving the sensitivity of the assay. We use this approach to detect β-actin messenger RNA (mRNA) transcripts.

Paper Details

Date Published: 19 June 2003
PDF: 11 pages
Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); doi: 10.1117/12.488210
Show Author Affiliations
Peter M. Goodwin, Los Alamos National Lab. (United States)
Rhiannon L. Nolan, Los Alamos National Lab. (United States)
Hong Cai, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 4962:
Manipulation and Analysis of Biomolecules, Cells, and Tissues
Dan V. Nicolau; Joerg Enderlein; Robert C. Leif; Daniel L. Farkas, Editor(s)

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