This Bacterial Artificial Chromosome Resource proposal contains four main components: BAC library preparation, BAC library Characterization, BAC library distribution and Improvement of genomic DNA cloning technology. Six new human genomic BAC libraries with tenfold genome representation will be constructed during the first three years. The libraries, based on variants and improvements of current BAC technology, will complement and extend the repertoire of BAC libraries currently available for genome sequencing an analysis. The new clone collections will be analyzed by a standardized set of tests, including the probing with a large set of distributed genomic markers and by exploring library continuity for two single-megabase regions. In addition to the analysis of the genomic clones present in the BAC libraries, we will search for sequences absent in the BAC libraries using a """"""""genome versus BAC library"""""""" subtraction approach. This search will be based on the representational difference analysis (RDA) technology developed by Lisitsyn and Wigler. Sequences developed by RDA will be used as markers to screen and compare BAC libraries developed by variant cloning approaches. All conventional BAC libraries developed during the first three years and later clone resources prepared during years 4 and 5, will be widely distributed through the scientific community requesting only reimbursement for duplication and distributions costs. To improve the cloning of genomic DNA sequences, we will explore new vectors, different restriction digests, other E.coli cloning hosts, improved DNA handling technologies and alternative ways of E.coli transformation. The use of the targeted recombination cloning approach (""""""""TAR"""""""") developed by Larionov and coworkers, is anticipated as an option to re-clone large genomic segments from the same or different haplotype. We will facilitate recombinational re-cloning by appropriate BAC vector modification, resulting in """"""""TAR-BAC"""""""" vectors. Two subcontracts will supply expertise not present in our group, with respect to magnetic separation and concentration of DNA, and Oral packaging/transformation based on the bacteriophage T4 system. Library information and technology improvements will be disseminated through publication and through our home page at the World Wide Web (http://bacpac.med.buffalo. edu)
