Skip to main content
SpringerLink
Log in

Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Enzymatic ligation assisted by nuclease (ELAN) reaction scheme and results.

Similar content being viewed by others

References

  1. Cost, G.J. & Cozzarelli, N.R. Directed assembly of DNA molecules via simultaneous ligation and digestion. Biotechniques 42, 84–89 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Bustamante, C., Bryant, Z. & Smith, S.B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).

    Article  PubMed  Google Scholar 

  3. 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).

    Article  CAS  PubMed  Google Scholar 

Download references

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).

Author information

Author notes

  1. Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Current address: Sangamo BioSciences, 501 Canal Boulevard, Richmond, California 94804, USA.

Authors
  1. Gregory J Cost
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and permissions

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.325

  • Springer Nature Limited

This article is cited by

Search

Navigation