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Targeted screening for induced mutations

Nature Biotechnology volume 18, pages 455–457 (2000)Cite this article

Abstract

With the accumulation of large-scale sequence data, emphasis in genomics has shifted from determining gene structure to testing gene function, and this relies on reverse genetic methodology. Here we explore the feasibility of screening for chemically induced mutations in target sequences in Arabidopsis thaliana. Our TILLING (Targeting Induced Local Lesions IN Genomes) method combines the efficiency of ethyl methanesulfonate (EMS)-induced mutagenesis1 with the ability of denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis2. Importantly, this method generates a wide range of mutant alleles, is fast and automatable, and is applicable to any organism that can be chemically mutagenized.

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Figure 1: (A) Diagrammatic structure of A. thaliana chromomethylase genes.

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Acknowledgements

We thank Bruce Draper for helpful discussions. This work was supported by grant RO1 GM29009 (to S.H.) from the National Institutes of Health. S.H. is an investigator of the Howard Hughes Medical Foundation, which also provided support for Karen Wolfe of the James Roberts lab, whom we thank for helping us with the screen.

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Authors and Affiliations

  1. Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, 98109-1024, WA

    Claire M. McCallum, Elizabeth A. Greene & Steven Henikoff

  2. Molecular and Cellular Biology Program, University of Washington, Seattle, 98199, WA

    Claire M. McCallum

  3. Department of Botany, University of Washington, Seattle, 98199, WA

    Luca Comai

  4. Howard Hughes Medical Institute Research Laboratories, Fred Hutchinson Cancer Research Center, Seattle, 98109, WA

    Steven Henikoff

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  1. Claire M. McCallum
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  2. Luca Comai
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  4. Steven Henikoff
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Correspondence to Steven Henikoff.

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McCallum, C., Comai, L., Greene, E. et al. Targeted screening for induced mutations. Nat Biotechnol 18, 455–457 (2000). https://doi.org/10.1038/74542

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