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.
This is a preview of subscription content, access via your institution
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Cost, G.J. & Cozzarelli, N.R. Directed assembly of DNA molecules via simultaneous ligation and digestion. Biotechniques 42, 84–89 (2007).
Bustamante, C., Bryant, Z. & Smith, S.B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).
Kodumal, S.J. et al. Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster. Proc. Natl. Acad. Sci. USA 101, 15573–15578 (2004).
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).
The author declares no competing financial interests.
About this article
Cite this article
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
Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture
Nature Genetics (2018)
TFIIH generates a six-base-pair open complex during RNAP II transcription initiation and start-site scanning
Nature Structural & Molecular Biology (2017)
Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression
Molecular Therapy (2016)