What steps are required to produce a validated ZFN?
• Using our proprietary algorithm, we design several candidate ZFN molecules in-silico. • We then assemble the candidate ZFNs for testing. • The assembled ZFNs are then tested for DNA binding specificity and for the ability to cleave at the target site on the chromosome in a cellular assay (e.g. the mismatch assay described here). • We then select the best ZFN pair using the above criteria and ship that to our customers. How is the binding specificity of the ZFN tested? The binding specificity of the zinc finger modules in the Sangamo archive have previously been characterized by a number of in-vitro methods (ELISA among others). How has the cleavage ability of my ZFN been tested? To test the cleavage ability of the ZFNs we use a mismatch-specific nuclease assay. In this assay, PCR primers are used to amplify across the ZFN cleavage site in genomic DNA extracted from ZFN-treated cells. The resulting PCR products, which are now a mixture of wild type and mutated amplicons, are then dena
