title: Pairwise selection assembly for sequence-independent construction of long-length DNA.
abstract: 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 approximately 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. 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.
authors.affiliation: Codon Devices, Inc., One Kendall Square, Building 300, Cambridge, MA 02139, USA. wblake@glbiosciences.com
authors.forename: William J
authors.initials: WJ
authors.lastname: Blake
authors.forename: Brad A
authors.initials: BA
authors.lastname: Chapman
authors.forename: Anuradha
authors.initials: A
authors.lastname: Zindal
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lee
authors.forename: Shaun M
authors.initials: SM
authors.lastname: Lippow
authors.forename: Brian M
authors.initials: BM
authors.lastname: Baynes
mesh_terms: D001483:Base Sequence; D015825:Chromosomes, Fungal; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D005818:Genetic Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D004247:DNA