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Fluorescence Detection of Single DNA Molecules

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Abstract

Single-molecule detection (SMD) and single-molecule fluorescence resonance energy transfer (smFRET) were conducted using Cy3- and Cy5-labeled single-strand DNAs (ssDNAs) either immobilized on substrates or encapsulated in microdroplets. High-quality fluorescent images were obtained using a total internal reflection fluorescence microscope (TIRFM). In the substrate system, deposition of a low concentration of fluorescence molecules on substrates through electrostatic adsorption showed that most of the fluorescence spots were single molecules, and the mean value of signal to noise ratio (S/N) reached 6.9 ± 0.34. smFRET analysis was conducted through immobilization of donor- and acceptor-labeled oligonucleotides on substrates. In the droplet system, fluorophor-labeled oligonucleotides were injected into T-type microfluidics. Single and double fluorophor-labeled DNA molecules encapsulated in droplets were detected, the FRET efficiency and inter-dye distance of a single donor-acceptor pair were measured accurately. smFRET was conducted detailedly in the tortuous channel for the first time.

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Acknowledgments

This work is supported by the State High-Tech Research and Development Plan (863) (grant No. 2012AA02A104) and Foundation for Innovation in Science and Technology, Shanghai Jiao Tong University.

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  1. School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China

    Weidong Huang, Yue Wang & Zhimin Wang

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  1. Weidong Huang
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Correspondence to Zhimin Wang.

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Huang, W., Wang, Y. & Wang, Z. Fluorescence Detection of Single DNA Molecules. J Fluoresc 25, 1267–1277 (2015). https://doi.org/10.1007/s10895-015-1615-0

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