Streptavidin-coated beads bind and remove the small biotinylated fragment from the digestion. The amplified oligos are then digested at high-temperature to expose the microbead barcode as a single-stranded DNA overhang. Upon arrival, each sub-pool is PCR amplified from the pool with a biotinylated primer. After the oligo design is completed, the library is ordered from a commercial oligo pool vendor. Typically, the library is split into batches of 384 genes, each with a unique pair of sub-pool amplification primers. All of the oligos needed to assemble one particular gene are given the same microbead barcode. Restriction sites and a microbead barcode are added to each oligo. Briefly, the genes to be synthesized are first bioinformatically split into several fragments, such that each fragment can fit on an oligo. We then break the emulsion and recover our library of assembled genes.Īn animation of DropSynth can be found at the bottom of this post. Then the barcodes are removed, and gene assembly takes place inside of those droplets by polymerase chain assembly (basically PCR). The beads are then emulsified, thereby isolating and concentrating the oligos into a picoliter-sized droplet. DropSynth overcomes this barrier by isolating individual assembly reactions in a vortexed emulsion, allowing for the entire gene library to be assembled in one pot.ĭropSynth uses a set of barcoded beads, such that each bead pulls down the required oligos for a particular gene’s assembly. Previous gene synthesis methods require isolating individual gene assemblies in different reactions, which becomes cost-prohibitive for assembling thousands of genes and requires expensive automation equipment. In the meantime, we’ve been asked questions by many others while presenting the work to colleagues, and thought we’d detail some of those answers below.ĭropSynth is a new gene synthesis method whereby large gene libraries are assembled from microarray-derived oligo libraries within water-in-oil droplets. We will gather useful documentation for the method at. DropSynth is a fairly straightforward protocol that should be easy for individual labs to perform with no specialized equipment. ![]() We expect this to be the first report of a method which we will continue to improve in scale, cost, length, and ease over the coming years. In the published work, we build thousands of homologs of two essential bacterial genes from oligo arrays (~450-675bp in length), and then characterize homologs of the essential enzyme PPAT for their ability to complement in E. Briefly, DropSynth is a simple, low-cost method to build thousands of genes from microarray-derived oligos in a single reaction. We are excited today to release DropSynth to the world in a new paper published online in Science Magazine.
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