Zinc finger nucleases (ZFNs) are custom restriction enzymes that can be designed to recognize and cleave DNA in virtually any specific user-defined location. In my thesis research, I took advantage of the unique DNA-recognition properties of ZFNs to develop novel approaches for molecular cloning and plant genome engineering. First, I described expression and purification methods for ZFN10, a novel custom restriction endonuclease, and characterized its biochemical properties. I demonstrated that Ni-affinity purification was sufficient to produce a cloning-grade enzyme and that ZFN10 was tolerant to a range of target-site substitutions which could be predicted from specificities of recognition helices incorporated into the structure of its zinc finger protein (ZFP) domain. Second, I showed that ZFNs can be used for standard molecular cloning applications along with known type II restriction enzymes, and that because of their long target sites and design flexibility, they can accomplish such challenging tasks as cloning very large DNA fragments and custom-cloning native DNA molecules. Third, I developed a comprehensive method for ZFN testing, which can be used by research groups wishing to adopt ZFN technology for plant gene targeting. The testing consists of an in-vitro assay involving expression of a ZFN in E. coli with subsequent use of the bacterial lysate to digest a plasmid carrying the ZFN target site, and of a series of plant assays based on reconstruction of a GUS reporter's expression following transient or stable delivery of a mutated GUS-encoding gene and ZFN-expressing cassettes into target plant cells. This work expands the current collection of restriction endonucleases used in molecular cloning with potentially hundreds of thousands of new enzymes and provides the foundation for widespread use of ZFNs for targeted modification of plant genomes.
|School:||State University of New York at Stony Brook|
|School Location:||United States -- New York|
|Source:||DAI-B 71/02, Dissertation Abstracts International|
|Keywords:||DNA engineering, Gene targeting, Molecular cloning, Nucleases, Zinc finger|
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