Separation of Cleaved Products
The first paper describing TILLING used dHPLC 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 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.
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