title: Positive selection improves the efficiency of DNA assembly.
abstract: With the advent of synthetic biology and cell engineering, the demand for large synthetic DNA fragments has been steadily increasing. Consequently, a number of multi-fragment cloning technologies optimized for the assembly of sizable DNA constructs have been developed. Still, screening for the right clone can be tedious because the high incidence of illegitimate assembly results in a relatively large proportion of missing or shuffled DNA elements. To mitigate this risk, we have developed a strategy that reduces the rate of fragment mis-assembly and is compatible with a variety of cloning methodologies. The approach is based on the positive selection of truncated plasmid markers, which are rendered active by providing their missing sequences during the assembly process. The method has been successfully validated in the context of complex in vivo and in vitro homologous recombination workflows, but it could be readily adapted to other cloning strategies, including those based on restriction endonucleases.
authors: Thermo Fisher Scientific, Carlsbad, CA 92008, USA.
authors: Chang-Ho
authors: CH
authors: Baek
authors: Thermo Fisher Scientific, Carlsbad, CA 92008, USA.
authors: Jonathan
authors: J
authors: Chesnut
authors: Thermo Fisher Scientific, Carlsbad, CA 92008, USA. Electronic address: federico.katzen@thermofisher.com.
authors: Federico
authors: F
authors: Katzen
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D004247:DNA
keywords: Cloning; DNA assembly; Positive selection; Synthetic biology