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

1email, 1email, 1email, 2email, 3email, 4email, 2email, 1email and 1,* email
Received: 9 December 2013; in revised form: 25 February 2014 / Accepted: 7 March 2014 / Published: 12 March 2014
(This article belongs to the Special Issue Single Biomolecule Detection)
View Full-Text   |   Download PDF [383 KB, uploaded 21 June 2014]
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
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Export to BibTeX |
EndNote


MDPI and ACS Style

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.

AMA Style

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(3):5174-5182.

Chicago/Turabian Style

Takahashi, Shunsuke; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji. 2014. "A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A." Sensors 14, no. 3: 5174-5182.


Sensors EISSN 1424-8220 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert