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Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

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Abstract

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.

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Acknowledgment

The authors wish to express their gratitude to Dr. B.-H. Chung at Korean Research Institute of Bioscience and Biotechnology (KRIBB) for his assistance with the Dual-View system. This work was supported by a grant from the Basic Research Program of the Korea Science & Engineering Foundation (R01-2004-000-10056-0).

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Authors and Affiliations

  1. Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju, 561-756, South Korea

    Seong Ho Kang & Yun-Jeong Kim

  2. Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA, 50011, USA

    Edward S. Yeung

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  1. Seong Ho Kang
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  2. Yun-Jeong Kim
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  3. Edward S. Yeung
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Correspondence to Seong Ho Kang.

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Kang, S.H., Kim, YJ. & Yeung, E.S. Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy. Anal Bioanal Chem 387, 2663–2671 (2007). https://doi.org/10.1007/s00216-007-1134-1

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  • DOI: https://doi.org/10.1007/s00216-007-1134-1

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