Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy.
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.
Kang
Seong Ho
SH
Department of Chemistry and Research Institute of Physics and Chemistry (RINPAC), Chonbuk National University, Jeonju 561-756, South Korea. shkang@chonbuk.ac.kr
Kim
Yun-Jeong
YJ
Yeung
Edward S
ES
D001483:Base Sequence; D003116:Color; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D004247:DNA