High-throughput TILLING for Arabidopsis.
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.
Till
Bradley J
BJ
Basic Sciences Division, Fred Hutchison Cancer Research Center, Department of Biology, University of Washington, Seattle, USA.
Colbert
Trenton
T
Codomo
Christine
C
Enns
Linda
L
Johnson
Jessica
J
Reynolds
Steven H
SH
Henikoff
Jorja G
JG
Greene
Elizabeth A
EA
Steine
Michael N
MN
Comai
Luca
L
Henikoff
Steven
S
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
D017931:DNA Primers; D018744:DNA, Plant; D005456:Fluorescent Dyes