Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection

Peter M. Goodwin; Rhiannon L. Nolan; Hong Cai

doi:10.1117/12.488210

19 June 2003 Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection

Peter M. Goodwin, Rhiannon L. Nolan, Hong Cai

Author Affiliations +

Proceedings Volume 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues; (2003) https://doi.org/10.1117/12.488210
Event: Biomedical Optics, 2003, San Jose, CA, United States

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.

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Peter M. Goodwin, Rhiannon L. Nolan, and Hong Cai "Quantitative analysis of specific nucleic acid sequences by two-color single-molecule fluorescence detection", Proc. SPIE 4962, Manipulation and Analysis of Biomolecules, Cells, and Tissues, (19 June 2003); https://doi.org/10.1117/12.488210

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