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Sensors 2014, 14(3), 5174-5182; doi:10.3390/s140305174
Article

A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

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1 Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan 2 Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 4418580, Japan 3 Cellular Dynamics Laboratory, RIKEN, Saitama 3510198, Japan 4 Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 9838551, Japan
* Author to whom correspondence should be addressed.
Received: 9 December 2013 / Revised: 25 February 2014 / Accepted: 7 March 2014 / Published: 12 March 2014
(This article belongs to the Special Issue Single Biomolecule Detection)
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Abstract

Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3’-5’exo–) was promoted by DNA stretching with buffer flow.
Keywords: single-molecule observation; single-stranded DNA; replication protein A (RPA); DNA polymerase; DNA synthesis single-molecule observation; single-stranded DNA; replication protein A (RPA); DNA polymerase; DNA synthesis
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
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Takahashi, S.; Kawasaki, S.; Miyata, H.; Kurita, H.; Mizuno, T.; Matsuura, S.-I.; Mizuno, A.; Oshige, M.; Katsura, S. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A. Sensors 2014, 14, 5174-5182.

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