Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.
CITATION STYLE
Till, B. J., Colbert, T., Tompa, R., Enns, L. C., Codomo, C. A., Johnson, J. E., … Henikoff, S. (2003). High-throughput TILLING for functional genomics. Methods in Molecular Biology (Clifton, N.J.), 236, 205–220. https://doi.org/10.1385/1-59259-413-1:205
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