TILLING (molecular biology)
TILLING (Targeting Induced Local Lesions in Genomes) is a method in molecular biology that allows directed identification of mutations in a specific gene. TILLING was introduced in 2000, using the model plant Arabidopsis thaliana, and expanded on into other uses and methodologies by a small group of scientists including Luca Comai. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, maize, wheat, rice, soybean, tomato and lettuce. Overview The method combines a standard and efficient technique of mutagenesis using a chemical mutagen such as ethyl methanesulfonate (EMS) with a sensitive DNA screening-technique that identifies single base mutations (also called point mutations) in a target gene. The TILLING method relies on the formation of DNA heteroduplexes that are formed when multiple alleles are amplified by PCR and are then heated and slowly cooled. A “bubble” forms at the mismatch of the two DNA strands, which is then cleaved by a single stranded nuclease. The products are then separated by size on several different platforms (see below). Mismatches may be due to induced mutation, heterozygosity within an individual, or natural variation between individuals. EcoTILLING is a method that uses TILLING techniques to look for natural