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Methods Mol Biol. 2003;236:205-20.

High-throughput TILLING for functional genomics.

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1
BasicSciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

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. 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.

PMID:
14501067
DOI:
10.1385/1-59259-413-1:205
[Indexed for MEDLINE]
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