Research Project
3333472
Bacteriophage P1 has the capacity to package as much as 115 kb of DNA into its capsid. We have taken advantage of this property to develop a vector, and an in vitro packaging system, that permits the efficient cloning and amplification of segments of DNA that are as large as 100 kilobases (kb) in size, more than twice the cloning capacity of cosmid cloning systems. This proposal has the following five primary objectives: (1) To modify the P1 cloning vector to provide a direct selection for inserts and to flank the polylinker cloning site with T3 and T7 promoter elements; (2) To increase the efficiency of the packaging reaction and to minimize the contribution of small headed variants to that process. This will be accomplished by overproducing the P1 pac cleavage proteins and by purifying large P1 heads and P1 tails. These components will be used to optimize the various step in the packaging process; (3) To evaluate te fidelity with which large molecular weight DNA inserts are cloned; (4) To develop the P1 lox-Cre site-specific recombination system as means of efficiently mapping restriction enzyme sites on cloned inserts; and (5) To assess the potential utility of P1 processive headful packaging in rapidly mapping DNA segments as large as 0.5-1.0 Mb. The large DNA cloning capacity of the P1 system will permit the isolation in Escherichia coli (E. coli) of functional genes whose size is in the 40-100 kb range. Moreover, it will permit one to localize and map genes by chromosomal walking procedures at least twice as fast, and with greater accuracy, than is now possible with cosmid vectors.
