Abstract
This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4–16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.
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References
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Acknowledgements
This work was funded in part by the grant GM31657 to Nicholas R. Cozzarelli from the National Institutes of Health. G.J.C. was the Amgen Fellow of the Damon Runyon Cancer Research Foundation (grant 1702-02), and was supported by a Kirschstein National Research Service award from the National Institutes of Health (F32 GM075645-01).
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Cost, G. Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA. Nat Protoc 2, 2198–2202 (2007). https://doi.org/10.1038/nprot.2007.325
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DOI: https://doi.org/10.1038/nprot.2007.325
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