TILLING using a non-denaturing polyacrylamide detection method: A) Visualization of four-fold DNA pools digested with CJE after running on a non-denaturing 3% polyacrylamide gel for 75 minutes. Putative mutations in the pools are identified by the presence of two bands (indicated by white arrows) whose sizes add up to the full length PCR product. In pool 5, more than two bands are visible, representing two mutations within this pool (yellow arrows). Size markers (M) are included throughout the gel. This is a composite of four images whose contrast has been adjusted differently to allow better visualization. B) For each positive pool (labeled 1 through 7), the four individual DNAs (labeled a through d) are organized in a 96-well plate and used for PCR amplification of the target region. After PCR, paired pools are assembled by combining 6 μl of PCR product from two individuals and organizing them into a new 96-well plate. For example, row a+b contains 6 μl from individual a and 6 μl from individual b. C) Heteroduplexes are formed through denaturing and annealing of the pooled PCR products and mismatches were digested with CJE. Cleaved fragments were visualized using the non-denaturing polyacrylamide gel electrophoresis set-up as before. Each column is run in adjacent lanes, such that the first four lanes contain the four two-fold pools (a+b, c+d, a+c and b+d) from column 1. True mutations are replicated in two separate gel lanes within each set of four, producing a unique banding pattern (represented below each set of four lanes and represented in panel D). According to this pattern, the mutation can be unequivocally assigned to one of the individual DNAs. E) The PCR product from these individuals (leftover from the PCR on panel B) is sequenced and the identity of the mutation is determined.