As more genome projects shift into the large-scale sequencing phase, one of the overwhelming technical challenges remains in developing an efficient method for producing minimum tiling paths of sequence-ready clones across a region targeted for sequencing. A resource for accomplishing this task is the availability of deep coverage BAC libraries that have been characterized by end sequencing and restriction fingerprinting. We propose to initially construct BAC libraries using traditional methods, and then to aggressively integrate new cloning strategies. The first new technology we propose to incorporate will be the use of a BAC vector (pBAC/oriV) containing a second origin of replication (oriV), which is stringently regulated and may be induced to increase the plasmid copy number 50-fold. Libraries constructed from this vector will facilitate the processes of restriction fingerprinting, end sequencing, and shotgun library construction that will contribute to reducing the costs of these projects. We will investigate methods to produce random libraries based on physically sheared DNA and cloning into appropriately designed vectors or the random integration of vectors into genomic DNA. We will also investigate the use of phage systems for packaging of random DNA and subsequent increase of transfection versus electroporation. The phage systems also have the benefit of packaging discrete fragment sizes, thus producing libraries with a very tightly defined insert size range. Finally, we will produce E. coli host strains that are deficient in transposable elements to prevent transposition into cloned DNA. We will establish a BAC production and research facility that will be capable of producing high quality libraries and will distribute them to the research community in microtiter plates or as high density hybridization filters.
