A protocol for TILLING and Ecotilling in plants and animals

We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping ofinduced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also beenadapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes withina particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samplesare denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in theseheteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated usingdenaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicatesthe approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-wellliquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands ofsamples can be screened daily.INTRODUCTIONThe acquisition of large-scale nucleotide sequence data has led tothe introduction of methods that target the disruption of specificgenes, also known as reverse genetics. Although potentially verypowerful, reverse genetic approaches can be limited if the methodused to induce the genetic change is not generally applicable todifferent organisms, as is often the case for transgenic strategies.However, chemicals and other agents have been used for severaldecades to induce mutations in a variety of organisms, makingtraditional mutagenesis an attractive strategy for reverse genetics.Chemical mutagenesis is especially valuable for agricultural appli-cations because, unlike transgenic approaches, it is not hamperedby restrictive regulatory and consumer issues. In addition, muta-genesis-based methods for reverse genetic applications can providemore than just gene knockouts; mutagens such as ethyl methane-sulfonate primarily induce point mutations, providing allelic seriesthat also include useful missense mutations