BACTERIOPHAGE CLONING SYSTEM--HIGH MOLECULAR WEIGHT DNA

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During the past two and one-half years we have devoted much effort to improving the phage Pl cloning system. At the present time it is accepted as a reasonable alternative to cosmid and YAC systems for the cloning of high molecular weight (HMW) DNA. We have constructed Pl mouse and human libraries in our laboratory and have supported the efforts of others to construct rat and Drosophila libraries. In this proposal we wish to extend our studies in four directions: 1. We want to evaluate how faithful the Pl cloning process is and how representative the new Pl mouse and human libraries are. To do this we will determine whether the Pl system is able to clone regions of genomic DNA that are not clonable or are unstable in cosmid vectors. We also will initiate efforts to construct contigs in two regions of the mouse genome in collaboration with Drs. Gasser and Nadeau to determine how efficiently the Pl system can generate a long range physical map. 2. We want to increase the efficiency of Pl cloning and the purity of the reaction needed to package and recover cloned DNA. This will permit us to clone HMW DNA under less than optimal conditions (e.g. with DNA derived from sorted chromosomes) and will avoid problems associated with nuclease activity in the crude packaging extracts that compromise the efficiency of those extracts. 3. We wish to discover how the Pl system packages less than headful-sized (110 kb) DNA in large heads. Hopefully we can use that information to inhibit that reaction. 4. We want to continue efforts to develop transposon systems that will permit us to fragment any cloned insert in a controlled manner. This will be useful in localizing physical and functional determinants on those inserts and will permit their re-introduction into mammalian cells.