A protocol for TILLING and Ecotilling in plants and animals

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• Genetic variation is crucial for understanding organismal adaptation and disease.
• Efficient methods are needed to discover and map mutations within genomes.
• Reverse genetics provides a powerful approach to link genotype with phenotype.

Purpose of the Study:

• To introduce Targeting-Induced Local Lesions IN Genomes (TILLING) as a novel reverse-genetic strategy.
• To demonstrate the utility of TILLING for discovering and mapping induced mutations.
• To present Ecotilling as an adaptation for cataloguing natural polymorphisms.

Main Methods:

• Utilizes PCR with fluorescently labeled, gene-specific primers to amplify target DNA regions.
• Generates heteroduplexes from polymorphic DNA strands, followed by mismatch cleavage with a single-strand specific nuclease.
• Employs denaturing polyacrylamide gel electrophoresis and fluorescence detection for size fractionation and visualization of cleaved products.

Main Results:

• Cleaved product migration accurately indicates the approximate location of nucleotide polymorphisms.
• High-throughput screening is achieved through sample pooling, multi-well liquid handling, and automated gel band mapping.
• Thousands of genomic DNA samples can be screened daily, significantly increasing efficiency.

Conclusions:

• TILLING is a broadly applicable strategy for mutation discovery and mapping in mutagenized organisms.
• Ecotilling effectively facilitates the discovery and cataloguing of natural genetic variations.
• The described methods offer a cost-effective and high-throughput approach to genetic screening.