title: High-throughput TILLING for Arabidopsis.
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.
authors.affiliation: Basic Sciences Division, Fred Hutchison Cancer Research Center, Department of Biology, University of Washington, Seattle, USA.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Till
authors.forename: Trenton
authors.initials: T
authors.lastname: Colbert
authors.forename: Christine
authors.initials: C
authors.lastname: Codomo
authors.forename: Linda
authors.initials: L
authors.lastname: Enns
authors.forename: Jessica
authors.initials: J
authors.lastname: Johnson
authors.forename: Steven H
authors.initials: SH
authors.lastname: Reynolds
authors.forename: Jorja G
authors.initials: JG
authors.lastname: Henikoff
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Greene
authors.forename: Michael N
authors.initials: MN
authors.lastname: Steine
authors.forename: Luca
authors.initials: L
authors.lastname: Comai
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D018744:DNA, Plant; D005456:Fluorescent Dyes / Q000494:pharmacology; D017343:Genes, Plant; D005821:Genetic Techniques; D018745:Genome, Plant; D008957:Models, Genetic; D016296:Mutagenesis; D009154:Mutation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D017931:DNA Primers; D018744:DNA, Plant; D005456:Fluorescent Dyes