PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets.
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.
Kojima
Takaaki
T
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Takei
Yoshiaki
Y
Ohtsuka
Miharu
M
Kawarasaki
Yasuaki
Y
Yamane
Tsuneo
T
Nakano
Hideo
H
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004655:Emulsions; D005434:Flow Cytometry; D016680:Genome, Bacterial; D015698:Genomic Library; D023281:Genomics / Q000379:methods*; D008280:Magnetics; D008863:Microspheres; D009821:Oils / Q000737:chemistry; D010231:Paracoccus denitrificans / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004655:Emulsions; D009821:Oils; D012097:Repressor Proteins; D014157:Transcription Factors; D014867:Water; D004247:DNA