title: High-throughput, cost-effective verification of structural DNA assembly.
abstract: DNA 'assembly' from 'building blocks' remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼ 1 kb), pathway engineering (∼ 10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.
authors.affiliation: Amyris, Inc., 5885 Hollis Street, Suite 100, Emeryville, CA 94608, USA.
authors.forename: Yandi
authors.initials: Y
authors.lastname: Dharmadi
authors.forename: Kedar
authors.initials: K
authors.lastname: Patel
authors.forename: Elaine
authors.initials: E
authors.lastname: Shapland
authors.forename: Daniel
authors.initials: D
authors.lastname: Hollis
authors.forename: Todd
authors.initials: T
authors.lastname: Slaby
authors.forename: Nicole
authors.initials: N
authors.lastname: Klinkner
authors.forename: Jed
authors.initials: J
authors.lastname: Dean
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Chandran
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D019075:Electrophoresis, Capillary; D017422:Sequence Analysis, DNA; D058615:Synthetic Biology / Q000191:economics / Q000379:methods*
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes