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DNA cloning by homologous recombination in Escherichia coli

Nature Biotechnology volume 18, pages 1314–1317 (2000)Cite this article

Abstract

The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology. The pioneering work in the early 1970s, using DNA ligases to paste DNA into episomal vectors, is still the most widely used approach. Here we describe a different principle, using ET recombination1,2, for directed cloning and subcloning, which offers a variety of advantages. Most prominently, a chosen DNA region can be cloned from a complex mixture without prior isolation. Hence cloning by ET recombination resembles PCR in that both involve the amplification of a DNA region between two chosen points. We apply the strategy to subclone chosen DNA regions from several target molecules resident in E. coli hosts, and to clone chosen DNA regions from genomic DNA preparations. Here we analyze basic aspects of the approach and present several examples that illustrate its simplicity, flexibility, and remarkable efficiency.

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Figure 1: Subcloning by ET recombination.
Figure 2: Cloning by ET recombination.
Figure 3: Additional aspects involved in ET subcloning.

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Acknowledgements

The authors like to thank Michelle Meredyth and Inhua Muyrers-Chen for critical readings of the manuscript. This work was supported in part by grants from the Volkswagen Foundation, Program on Conditional Mutagenesis, and the NIH, National Institute for Aging. Y.Z. was a recipient of an EMBO fellowship.

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Authors and Affiliations

  1. Gene Expression Program, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany

    Youming Zhang, Joep P.P. Muyrers, Giuseppe Testa & A. Francis Stewart

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  1. Youming Zhang
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  2. Joep P.P. Muyrers
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  3. Giuseppe Testa
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Correspondence to A. Francis Stewart.

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Zhang, Y., Muyrers, J., Testa, G. et al. DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18, 1314–1317 (2000). https://doi.org/10.1038/82449

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