Universal System for Directed Nucleic Acid Cleavage

We will develop a novel system for directed nucleic acid cleavage. This system is based uponour discovery that the 5' nucleases of several thermostable DNA polymerases can be directed to cleaveas site-specific endonucleases. This system will be thermostable, specific, efficient and easilyautomatable, and will find broad application in the fields of nucleic acids-based research and diagnostics.In Phase I we will seek to overcome the most substantial limitation of this technology, its inefficiencyat cleaving long 5' arms. We will examine the effects of reaction constituents and conditions on theefficiency of cleavage of a well-characterized substrate, performing a comprehensive examination of theeffects of different salts, divalent cations, denaturants, buffer pH values, helicases and temperatures.We will also use mutagenesis to alter the protein and influence its interaction with the substratestructure. During Phase II we will optimize the required substrate complex formations and nucleaseinteractions to develop this technology into a generic, directed nucleic acid cleavage system for bothsingle and double stranded nucleic acids.