Discovery of chemically induced mutations by TILLING

The term functional genomics encompasses a number of different approaches aimed at determining gene function on a genome-wide scale. The application of these approaches is greatly facilitated by the utilisation of new, high-throughput technologies applicable to almost any organism. As an example, sequence alignment-based comparisons are used to identify homologous sequences between and within species, transcriptional profiling to determine gene expression patterns and interaction analyses to help elucidate pathways, networks and protein complexes. However, although these analyses are extremely useful to extrapolate important features of a novel gene from a biochemical or a molecular point of view, they are not very informative in the context of the functional complexity of a living organism. In order to overcome these limitations, different reverse-genetics approaches have been conceived. Nonetheless, the tools for reverse genetics are not always transferable from one organism to another or from model species to non-model ones because in most cases the main drawback is the lack of efficient technical protocols exploitable for the majority of the plant species. A novel, reverse-genetics approach that combines the advantages of point mutations provided by chemical mutagenesis, with the advantages of PCR-based mutational screening has been introduced recently under the name of TILLING (Targeting Induced Local Lesion IN Genomes; McCallum et al. 2000). From a technical standpoint, the first step of a TILLING assay is the PCR amplification of a target DNA fragment of interest from pooled DNAs of multiple mutant individuals. In sample pools, heteroduplexes with a mismatched base pair are formed between wild-type and mutated fragments by denaturing and reannealing PCR products (Fig 21.1). Heteroduplexes are cleaved by an endonuclease enzyme able to recognize the mismatch position. Cleaved products are then resolved using denaturing polyacrilamide gel or capillary electrophoresis. When a positive signal is identified, individual DNA samples of the pools are mixed in equal amounts with the wild-type DNA and one-by-one reanalysed to identify the mutant individual plant; the induced mutations are eventually confirmed by sequencing. A detailed description of the technical aspects of the TILLING procedure is presented in the following section.