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

Sensors

Shunsuke Takahashi³  Shohei Kawasaki³  Hidefumi Miyata³  Hirofumi Kurita⁴  Takeshi Mizuno²  Shun-ichi Matsuura¹  Akira Mizuno⁴  Masahiko Oshige³ 

[1] Research Center for Compact Chemical System, National Institute of Advanced Industrial Science and Technology (AIST), Miyagi 9838551, Japan; E-Mail:;Cellular Dynamics Laboratory, RIKEN, Saitama 3510198, Japan; E-Mail:;Department of Chemical and Environmental Engineering, Graduate School of Engineering, Gunma University, Gunma 3768515, Japan; E-Mails:;Department of Environmental and Life Sciences, Graduate School of Engineering, Toyohashi University of Technology, Aichi 4418580, Japan; E-Mails:

关键词: single-molecule observation;    single-stranded DNA;    replication protein A (RPA);    DNA polymerase;    DNA synthesis;   

DOI  :  10.3390/s140305174

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2014 by the authors; licensee MDPI, Basel, Switzerland.