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Cited by 245

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Rapid and Reliable DNA Assembly via Ligase Cycling Reaction

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.·2014·ACS Synthetic Biology
DOI 10.1021/sb4001992
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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.

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Metadata

Authors
Kok, S. D., Stanton, L. H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., Platt, D., Shapland, E. B., Serber, Z., Dean, J., Newman, J. D., & Chandran, S. S.
Venue
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Published
2014-01-15
Volume
3
Issue
2
Pages
97-106
ISSN
2161-5063 (ISSN-L)
DOI
10.1021/sb4001992
OpenAlex
W2106022808
Cited by
245
References
1
FWCI
13.02
Type
journal article
Language
en
Open access
closed
Funders
Defence Research and Development Organisation (HR001-12-3-0006)

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