High-throughput TILLING for functional genomics.
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.
D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics*; D005821:Genetic Techniques; D016296:Mutagenesis / Q000235:genetics*; D009153:Mutagens / Q000494:pharmacology; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D010944:Plants / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D018744:DNA, Plant; D009153:Mutagens; D009692:Nucleic Acid Heteroduplexes