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  • Protocol Extension
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Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast

Nature Protocols volume 15, pages 734–749 (2020)Cite this article

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Abstract

Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR–Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.

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Fig. 1: Construction of the TAR vector with targeting sequences.
Fig. 2: TAR cloning of genomic regions lacking ARS sequences from microbial genomes.

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Acknowledgements

This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, USA.

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Authors and Affiliations

  1. Developmental Therapeutics Branch, National Cancer Institute (NCI), National Institutes of Health, Bethesda, MD, USA

    Natalay Kouprina, Vladimir N. Noskov & Vladimir Larionov

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  1. Natalay Kouprina
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  2. Vladimir N. Noskov
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  3. Vladimir Larionov
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Contributions

V.L., V.N.N. and N.K. designed the research and wrote the manuscript.

Corresponding author

Correspondence to Natalay Kouprina.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Yinhua Lu and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Kouprina, N. and Larionov, V. Mol. Ther. Methods Clin. Dev. 14, 16–26 (2019): https://doi.org/10.1016/j.omtm.2019.05.006

Lee, N. C. O., Larionov, V. and Kouprina, N. Nucleic Acids Res. 43, e55 (2015): https://doi.org/10.1093/nar/gkv112

Kouprina, N. and Larionov, V. Nat. Protoc. 3, 371–377 (2008): https://doi.org/10.1038/nprot.2008.5

Noskov, V. N. et al. BMC Genomics 4, 16 (2003): https://doi.org/10.1186/1471-2164-4-16

This protocol is an extension to: Nat. Protoc. doi:10.1038/nprot.2008.5.

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Kouprina, N., Noskov, V.N. & Larionov, V. Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast. Nat Protoc 15, 734–749 (2020). https://doi.org/10.1038/s41596-019-0280-1

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