IVC was initially developed as a mean of linking genotype to phenotype in a manner similar to living cells. Coalescence of droplets, and the exchange of high-molecular weight components (e.g., genes and proteins) between droplets, are usually negligible. Emulsions are highly stable for many days, if not months, even at temperatures close to 100☌. Making and breaking of emulsions takes 5-10 minutes and requires only basic laboratory equipment.Ĥ. Droplets of 1-50µm diameter can be generated. Complex biochemical processes such as DNA replication, gene transcription and translation, as well as simpler enzymatic or binding processes, can be performed within the aqueous compartments with a comparable efficiency to a bulk solution.ģ. The internal phase is biochemically active. Thus, the entire volume of the aqueous reaction mixture is compartmentalized and there is no need to remove or inactivate the external phase.Ģ. The external phase is biochemically inert. Water-in-oil emulsions afford several other distinct advantages:ġ. This concentration is high enough to drive transcription and translation from a single gene, and the ample detection of a single enzyme molecule. In such volumes, a single DNA or protein molecule is at a concentration of ~0.5nM. Emulsions routinely contain droplets with mean diameters of 2-3µm and volumes around 10 fl (10×10 -15 liter). The most distinct feature of IVC is the volume of the aqueous droplets, that is orders of magnitude smaller than in any other miniaturized assay format. These droplets provide a facile means of compartmentalizing reactions - 1 milliliter of emulsion contains >10 10 droplets, each of which serves as a discrete reaction vessel. In vitro compartmentalization (IVC) is a newly developed technology that uses the aqueous droplets of water-in-oil (w/o) emulsions as cell-like compartments. These demands are general and do not depend on the type of molecules (genes, proteins, small molecules, etc.) or activity (enzymatic, binding, inhibitory, etc.) that are being screened for, nor on the target of screening (functional genomics, directed evolution, drug discovery, etc.).Ĭonventional HTS approaches use either robotic 2D-arrays (e.g., microtitre plates), or living cells. Miniaturization, which has become the hallmark of modern science and technology, has also been applied to screening, thus leading to a variety of high-throughput screening (HTS) technologies that aim at the smallest possible reaction volumes and the most sensitive and rapid means of detection. All screening approaches rely on ways of compartmentalizing assay reactions, and means of rapidly screening various molecules imbedded in these compartments.
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