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Nucleic Acids Research 2005 33(17):e150; doi:10.1093/nar/gni143
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Published online 6 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets

Takaaki Kojima, Yoshiaki Takei, Miharu Ohtsuka, Yasuaki Kawarasaki, Tsuneo Yamane and Hideo Nakano*

Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

*To whom correspondence should be addressed. Tel: +81 52 789 4142; Fax: +81 52 789 4145; Email: hnakano{at}agr.nagoya-u.ac.jp

Received July 19, 2005. Revised September 13, 2005. Accepted September 13, 2005.

We have developed a novel method of genetic library construction on magnetic microbeads based on solid-phase single-molecule PCR in a fine and robust water-phase compartment formed in water-in-oil (w/o) emulsions. In this method, critically diluted DNA fragments were distributed over the emulsion as templates, where beads crosslinked with multiple primers and other PCR components were encapsulated to form multiple reaction compartments. The delivered DNA was then amplified and covalently immobilized on the beads in parallel, within individual compartments, to construct a genetic library on beads (GLOBE), which was readily applicable to a genomewide global scanning of genetic elements recognized by a defined DNA-binding protein. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. As a result of flow cytometry screening with an anti-His fluorescent antibody, the PhaR target fragments were enriched 1200-fold from this library with this system. Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.

Present address: Tsuneo Yamane, Chubu University, Matsumoto-cho, Kasugai, Aichi 487-8501, Japan


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