title: Rapid and reliable DNA assembly via ligase cycling reaction.
abstract: Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.
authors.forename: Stefan
authors.initials: S
authors.lastname: de Kok
authors.forename: Leslie H
authors.initials: LH
authors.lastname: Stanton
authors.forename: Todd
authors.initials: T
authors.lastname: Slaby
authors.forename: Maxime
authors.initials: M
authors.lastname: Durot
authors.forename: Victor F
authors.initials: VF
authors.lastname: Holmes
authors.forename: Kedar G
authors.initials: KG
authors.lastname: Patel
authors.forename: Darren
authors.initials: D
authors.lastname: Platt
authors.forename: Elaine B
authors.initials: EB
authors.lastname: Shapland
authors.forename: Zach
authors.initials: Z
authors.lastname: Serber
authors.forename: Jed
authors.initials: J
authors.lastname: Dean
authors.forename: Jack D
authors.initials: JD
authors.lastname: Newman
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Chandran
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D017353:Gene Deletion; D059765:Homologous Recombination; D016254:Mutagenesis, Insertional; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012441:Saccharomyces cerevisiae / Q000378:metabolism
chemical_list: D004247:DNA; D011088:DNA Ligases