Two cosmid cloning and vectors systems will be constructed and tested in Phase II of this SBIR. Both of these systems use in vivo site specific recombination system to streamline various methods of gene analysis. Specifically, the vectors have been designed to facilitate the types of analysis, which include restriction mapping and sequencing of large region (> 1Mbp) of the genome, that will be required to continue the rapid advances in molecular genetics. The first vector system enhances the jumping vectors constructed in Phase 1 to facilitate the construction of micro-jumping libraries of human jumping clones form hybrid human/hamster cell lines, in addition, methods will be developed to restrict map 10 Mbp regions of the gene. The second vector system facilities the construction of an ordered set of sequencing templates from a 40 Kbp DNA fragment in the cosmid vector pCS1. The resulting clones can be sequenced by currently available automated sequencing methods. We estimate the sequence templates can be constructed form cosmid clones for less than $0.05/bp. Some of the methods that will be implemented in these cloning systems are unique but research completed in Phase I of the SBIR grant has addressed the feasibility of the components of the systems.
