Abstract
The yeast one-hybrid (Y1H) system has been among the methods of choice to detect protein–DNA interactions. However, conventional Y1H systems with a single auxotrophic reporter gene often suffer from high incidence of false positives to demonstrate a limited power in large-scale screenings. Here we describe a refined Y1H system that uses two independent bait sequences, each controlling a distinct reporter gene integrated in the host genome. With these modifications and a method of targeted DNA methylation, we succeeded in efficient isolation of clones for methylated DNA-binding proteins from mammalian cDNA libraries.
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Acknowledgment
We thank Kazuyuki Mizushima for his contribution to vector construction.
This work was supported by Genome Network Project and Cell Innovation Project from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T.I.).
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Ota, K., Feng, SY., Ito, T. (2014). Detecting Protein–DNA Interactions Using a Modified Yeast One-Hybrid System. In: Miyamoto-Sato, E., Ohashi, H., Sasaki, H., Nishikawa, Ji., Yanagawa, H. (eds) Transcription Factor Regulatory Networks. Methods in Molecular Biology, vol 1164. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0805-9_5
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DOI: https://doi.org/10.1007/978-1-4939-0805-9_5
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