High Fidelity Linear MicroVector to Clone Complex Problematic and Large DNAs

The goal of this research is to dramatically improve the ability to clone and analyze large and unstable DNA fragments We propose to develop a novel linear cloning vector to maximize stability of cloned DNA in the bacterial host The vector will use the replication proteins of the phage Phi to achieve the highest accuracy of replication for all DNA inserts including AT rich repetitive or structurally unstable genes The cloning method will be quick and easy for DNA of any size range kb only requiring ligation of small vector arms to the DNA followed by transformation of bacteria This system will surpass existing standards for stability fidelity and lack of bias exceeding the limits of our previously developed pJAZZ linear cloning vector Importantly it will enable straightforward cloning of fosmid or BAC sized DNAs The vector will have minimal bacterial sequences providing significant benefits for mammalian cell transfection therapeutic protein production stem cell research and ultimately gene therapy The cloned DNAs will be bound to nuclear localization signals for enhanced delivery and expression in mammalian cells This Phi vector is urgently needed to help discover treatments for human illness For example the genome of the human malaria parasite Plasmodium falciparum is among the most difficult genomes to clone due to its repetitive highly AT rich nature We will collaborate with the Wellcome Trust Sanger Institute to construct and sequence long insert genomic libraries of Plasmodium falciparum This unique resource is critically needed to enable genetic research on this deadly pathogen The goal of this research is to greatly improve the ability to capture and analyze large unstable DNAs To demonstrate the usefulness of this system it will be used to clone large segments of the human malarial parasite Plasmodium falciparum an achievement that is currently impossible