Figure 1.

Cel-I-based Tilling strategy. We prepared a genomic DNA library from the mutagenized b pr cn sca/CyO, hb-lacZ fly lines and performed a three-step (1,2,3) nested PCR reaction (A) for a particular gene of interest. The third PCR reactions included the IRDye700 (red) and IRDye800 (blue) fluorophores bound to the forward and reverse primers, thereby differentially labeling the two ends of the amplicons (B). Then we denatured and reannealed the fragments to generate balancer/mutant heteroduplexes (C) that were digested by Cel-I (D). When a SNP is present, cleavage of the heteroduplexes generates two fragments labeled by IRDye700 and IRDye800, respectively, which can be detected upon denaturing polyacrylamide gel electrophoresis in a LI-COR setup. A typical LI-COR signal shows two fragments with complementary sizes that must sum up the original amplicon size. Subsequently, we sequenced the PCR products to confirm the molecular lesions (see Methods).