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Nucleic Acids Res. 2010 May;38(8):2594-602. doi: 10.1093/nar/gkq123. Epub 2010 Mar 1.

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

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1
Codon Devices, Inc., One Kendall Square, Building 300, Cambridge, MA 02139, USA. wblake@glbiosciences.com

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.

PMID:
20194119
PMCID:
PMC2860126
DOI:
10.1093/nar/gkq123
[Indexed for MEDLINE]
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