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. TILLING has since been used as a reverse genetics method in other organisms such as zebrafish, corn, 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 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 [1][2][3][4] is a method that uses TILLING techniques to look for natural mutations in individuals, usually for population genetics analysis. DEcoTILLING [5] 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[6] 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 add up to the full length product size. Advantages to the LICOR system are separation of large fragments (~ 2kb), high sample throughput (96 samples loaded on paper combs), and freeware to identify the mutations (GelBuddy). Drawbacks to the LICOR system is having to pour slab gels and long run times (~4 hours). TILLING and EcoTILLING methods are now being used on capillary systems from, Advanced Analytical Technologies, ABI and Beckman.

Several systems can be used to separate PCR products that are not labeled with dyes. Simple agarose electrophoresis systems will separate cleavage products inexpensively and with standard lab equipment. This was used to discover SNPs in chum salmon and was referred to as DEcoTILLING. The disadvantage of this system is reduced resolution compared to polyacrylamide systems. Elchrom Scientific sells Spreadex gels which are precast, can be high throughput and are more sensitive than standard polyacrylamide gels. Advanced Analytical Technologies Inc sells the AdvanCE FS96 dsDNA Fluorescent System which is a 96 capillary electrophoresis system that has several advantages over traditional methods; including ability to separate large fragments (up to 40kb), no desalting or precipitation step required, short run times (~30 minutes), sensitivity to 5pg/ul and no need for fluorescent labeled primers.

TILLING centers

Several TILLING centers exist over the world that focus on agriculturally important species:

References

  1. ↑ Comai, L.; Young, K.; Till, B. J.; Reynolds, S. H.; Greene, E. A.; Codomo, C. A.; Enns, L. C.; Johnson, J. E.; Burtner, C.; Odden, A. R.; Henikoff, S. (2004). "Efficient discovery of DNA polymorphisms in natural populations by Ecotilling". The Plant Journal. 37 (5): 778–786. PMID 14871304. doi:10.1111/j.0960-7412.2003.01999.x.
  2. ↑ Gilchrist, E. J.; Haughn, G. W.; Ying, C. C.; Otto, S. P.; Zhuang, J.; Cheung, D.; Hamberger, B.; Aboutorabi, F.; Kalynyak, T.; Johnson, L. E. E.; Bohlmann, J.; Ellis, B. E.; Douglas, C. J.; Cronk, Q. C. B. (2006). "Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa". Molecular Ecology. 15 (5): 1367–1378. PMID 16626459. doi:10.1111/j.1365-294X.2006.02885.x.
  3. ↑ Mejlhede, N.; Kyjovska, Z.; Backes, G.; Burhenne, K.; Rasmussen, S. K.; Jahoor, A. (2006). "EcoTILLING for the identification of allelic variation in the powdery mildew resistance genes mlo and Mla of barley". Plant Breeding. 125 (5): 461–467. doi:10.1111/j.1439-0523.2006.01226.x.
  4. ↑ Nieto, C.; Piron, F.; Dalmais, M.; Marco, C. F.; Moriones, E.; Gómez-Guillamón, M. L.; Truniger, V.; Gómez, P.; Garcia-Mas, J.; Aranda, M. A.; Bendahmane, A. (2007). "EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility". BMC Plant Biology. 7: 34. PMC 1914064 Freely accessible. PMID 17584936. doi:10.1186/1471-2229-7-34.
  5. ↑ Garvin, M. R.; Gharrett, A. J. (2007). "DEco-TILLING: An inexpensive method for single nucleotide polymorphism discovery that reduces ascertainment bias". Molecular Ecology Notes. 7 (5): 735–746. doi:10.1111/j.1471-8286.2007.01767.x.
  6. ↑ http://www.plantphysiol.org/content/156/3/1257.full

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