Rapid and Reliable DNA Assembly via Ligase Cycling Reaction

• Published in: ACS synthetic biology Vol. 3; no. 2; pp. 97 - 106
• Main Authors: Kok, Stefan de, Stanton, Leslie H, Slaby, Todd, Durot, Maxime, Holmes, Victor F, Patel, Kedar G, Platt, Darren, Shapland, Elaine B, Serber, Zach, Dean, Jed, Newman, Jack D, Chandran, Sunil S
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.02.2014
• Subjects: Cloning, Molecular; DNA - chemistry; DNA - metabolism; DNA Ligases - metabolism; Gene Deletion; Homologous Recombination; Mutagenesis, Insertional; Nucleic Acid Amplification Techniques - methods; Polymorphism, Single Nucleotide; Saccharomyces cerevisiae - metabolism; synthetic biology; chain-reaction cloning; high throughput; Gibson isothermal assembly; yeast homologous recombination; DNA assembly
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/sb4001992

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.