PMT2, fluorescence channel 2 (FITC) signal intensity. ( F) Typical histogram for beads containing P.denitrificans genomic library in FITC fluorescence intensity (for A-gated events). Alginate Microbeads are one of the most widely investigated cell encapsulation materials as they are biocompatible. Microfluidic droplet technologyhas the advantages of compartmentalizing reactions intodiscrete volumes, performing highly parallel reactions inmonodispersedroplets, reducing cross-contamination be-tween droplets, eliminating PCR bias and nonspecific am-plification, as well as enabling fast amplification with rapidthermocycling. Events gated in A (∼97% of total events) were subjected to the following analysis. ( E) Typical dot-plot of beads containing P.denitrificans genomic library in FS and SS. ( D) Typical histogram for beads containing Pp and negative-control fragments in FITC fluorescence intensity (for A-gated events). The polydimethylsiloxane (PDMS) microfluidic device is first treated using on-chip plasma-assisted deposition of polyvinyl alcohol, to selectively modify the hydrophobicity of microchannels. Events in region B were 51.5% of A-gated events. ( C) Histogram for ‘positive-control’ beads in FITC fluorescence intensity (for A-gated events). Events in region B were 1.6% of A-gated events. ( B) Histogram for ‘negative-control’ beads in FITC fluorescence intensity (for A-gated events). Events gated in A (∼80% of total events) were subjected to the following analysis. 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. ( A) Typical dot-plot for beads containing Pp and negative-control fragments in FS (forward scatter) and SS (side scatter). Therefore, this system is a powerful tool for analyzing the transcription network on a genomewide scale.įlow cytometry of bead–DNA–PhaR–antibody complexes. 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. We constructed a GLOBE of Paracoccus denitrificans and selected gene beads that were bound to the His-tagged transcription factor PhaR by flow cytometry. ![]() 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. 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. 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.
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