Pairwise selection assembly for sequence-independent construction of long-length DNA

William J. Blake, Brad A. Chapman, Anuradha Zindal, Michael E. Lee, Shaun M. Lippow, Brian M. Baynes
2010 Nucleic Acids Research  
The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than $10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations.
more » ... In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae.
doi:10.1093/nar/gkq123 pmid:20194119 pmcid:PMC2860126 fatcat:ht6w3yx3urdabkbfqpeugqq3am