Targeted screening for induced mutations

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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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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