![]() 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 or 1536 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.Ī visual description of DropSynth can be seen in the video above. 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. FAQ What is DropSynth?ĭropSynth is a new gene synthesis method whereby large gene libraries are assembled from microarray-derived oligo libraries within water-in-oil droplets. Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes. Plesa C*, Sidore AM*, Lubock N, Zhang D, Kosuri S. DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions bioRxiv, DOI: 10.1101/740977 (2019) Sidore AM*, Plesa C*, Samson JA, Kosuri S. ![]() 2020 - Multiplexed Characterization of Protein Families with DropSynth The video below provides a summary of how the DropSynth process works.ĭropSynth Summary from Calin Plesa on Vimeo. This website provides resources for scientists who wish to use DropSynth to assemble their own gene libraries. DropSynth was invented in the Kosuri Lab at UCLA and is now being developed in the Plesa Lab at UOregon. DropSynth is a simple, low-cost method to build thousands of genes from microarray-derived oligos in a single reaction.
0 Comments
Leave a Reply. |