TILLING (molecular Biology) - Overview

Overview

The method combines a standard and efficient technique of mutagenesis with 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 nucleases. 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 mutations in individuals, usually for population genetics analysis. DEcoTILLING is a modification of TILLING and EcoTILLING which uses an inexpensive method to identify fragments. Since the advent of NGS sequencing technologies, TILLING-by-sequencing has been developed based on Illumina sequencing of target genes amplified from multidimensionally pooled templates to identify possible single-nucleotide changes.