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