Abstract
Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, 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.
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References
McCallum, C. M., Comai, L., Greene, E. A., and Henikoff, S. (2000) Targeted screening for induced mutations. Nat. Biotechnol. 18, 455–457.
McCallum, C. M., Comai, L., Greene, E. A., and Henikoff, S. (2000) Targeting induced local lesions in genomes (TILLING) for plant functional genomics. Plant Physiol. 123, 439–442.
Colbert, T., Till, B. J., Tompa, R., et al. (2001) High-throughput screening for induced point mutations. Plant Physiol. 126, 480–484.
Till, B. J., Reynolds, S. H., Greene, E. A., et al. (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res. 13, 524–530.
Middendorf, L. R., Bruce, J. C., Bruce, R. C., et al. (1992) Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus. Electrophoresis 13, 487–494.
Greene, E. A., Codomo, C. A., Taylor, N. E., et al. (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164, 731–740.
Till, B. J., Colbert, T., Tompa, R., et al. (2003) High-throughput TILLING for functional genomics. Methods Mol. Biol. 236, 205–220.
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© 2006 Humana Press Inc.
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Till, B.J. et al. (2006). High-Throughput TILLING for Arabidopsis . In: Salinas, J., Sanchez-Serrano, J.J. (eds) Arabidopsis Protocols. Methods in Molecular Biology™, vol 323. Humana Press. https://doi.org/10.1385/1-59745-003-0:127
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DOI: https://doi.org/10.1385/1-59745-003-0:127
Publisher Name: Humana Press
Print ISBN: 978-1-58829-395-4
Online ISBN: 978-1-59745-003-4
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