Figure 1

Overview of the TILLING procedure. Pooled DNA is amplified using fluorescently tagged, gene-specific primers. The forward and reverse primers are labelled with different fluorophors that label both ends of the fragment. The amplified products are denatured by heating and then allowed to cool slowly so that they randomly re-anneal. Heteroduplex molecules form when mutant and wild-type PCR products anneal together, and these then become targets for a single-strand-specific nuclease found in Celery Juice Extract (CJE). The nuclease cleaves these heteroduplex fragments at one of the two strands, 3' to the site of the mismatch in the DNA. The PCR products that retain one of the labelled primers can then be detected on polyacrylamide denaturing LI-COR gels. Individuals with a mutation in the gene of interest are identified by the smaller cleavage fragment seen on the gel as well as the wild-type product. Because the nuclease cleaves either of the two strands randomly, cleavage products can be detected in both the IRD700 and IRD800 channels of the gel image. The position of the mutation within the PCR amplicon can be calculated from the size of the two fragments carrying the forward, IRD700-labeled primer, and the reverse, IRD800-labeled primer. Grey bands on the gel are thought to result from partial PCR products and aid in sizing of mutant bands.