The sheer size of the human genome is the major impediment to the construction of a comprehensive physical map. The need to rapidly clone, map, and manipulate large DNA molecules has become acute in conjunction with the Human Genome Project. Considerable successes have already been achieved with Yeast Artificial Chromosomes (YACs), and they are currently the major technology being employed in the mapping of the human genome. The main objective of this proposal is to investigate whether a new cloning system based on the E. coli F factor would be a valuable addition and/or alternative to the existing YAC cloning system. Preliminary results suggest that the F factor cloning system may offer advantages over the YAC cloning system in three important areas. First, it should be possible to prepare F factor libraries that contain very few chimeric clones. Second, it should be possible to stably propagate in F factor vectors certain DNA sequences that cause instability and rearrangements in YAC vectors. Third, the isolation and analysis of F factor clones should be substantially easier and faster. Adoption of the F factor cloning system would thus be expected to achieve significant savings of time and money associated with the mapping and analysis of complex genomes. Experiments are proposed to explicitly address these issues. To achieve these goals it will be necessary to develop specific protocols for the construction of large F factor libraries. In this respect it is significant that transformation frequencies of constructs in the 100-200 kbp range are some 100-fold higher in E. coli than in yeast. It should thus be possible to construct large F factor libraries in a single experiment. In addition, it should be possible to significantly improve the construction of libraries from very rare sources of DNA. Depending on the success of these investigations, the long-term objective of this proposal is to construct a representative human library in the F factor cloning system with 500 kbp average inserts and a 4- to 5-fold coverage of the human genome.
