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