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 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. Single strand cleavage enzymes There are several sources for single strand nucleases. The first widely used enzyme was mung bean nuclease, but this nuclease has been shown to have high non-specific activity, and only works at low pH, which can degrade PCR products and dye-labeled primers. The original source for single strand nuclease was from CEL1, or CJE (celery juice extract), but other products have entered the market including Frontier Genomics’ SNiPerase enzymes, which have been optimized for use on platforms that use labeled and unlabeled PCR products (see next section). Transgenomic isolated the single strand nuclease protein and sells it as a recombinant form. The advantage of the recombinant form is that unlike the enzyme mixtures, it does not contain non-specific nuclease activity, which can degrade the dyes on the PCR primers. The disadvantage is a substantially higher cost. Separation of cleaved products The first paper describing TILLING used HPLC to identify mutations (McCallum et al., 2000a). The method was made more high throughput by using the restriction enzyme Cel-I combined with the LICOR gel based system to identify mutations (Colbert et al.,2001). Advantages to using this system are that mutation sites can be easily confirmed and differentiated from noise. This is because different colored dyes can be used for the forward and reverse primers. Once the cleavage products have been run on a gel, it can be viewed in separate channels, and much like an RFLP, the fragment sizes within a lane in each channel should