High-throughput TILLING for functional genomics

Methods Mol Biol. 2003;236:205-20. doi: 10.1385/1-59259-413-1:205.

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.

MeSH terms

  • DNA, Plant / chemistry
  • DNA, Plant / genetics
  • Genes, Plant / genetics*
  • Genetic Techniques*
  • Mutagenesis / genetics*
  • Mutagens / pharmacology
  • Nucleic Acid Heteroduplexes / genetics
  • Plants / drug effects
  • Plants / genetics
  • Polymerase Chain Reaction / methods*

Substances

  • DNA, Plant
  • Mutagens
  • Nucleic Acid Heteroduplexes