A combination of mutations in bacteriophage X and its host Escherichia coli K-12 provides a convenient system for the isolation of large quantities of covalently closed circular DNA molecules. We describe two procedures for the large scale preparation of X DNA in the duplex circular form. In vitro analysis of the biochemical events involved in the metabolism of DNA requires DNA substrates of defined structure. The DNA of bacteriophage X is an especially useful substrate for such study for

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... l reasons: (i) it is small enough to be relatively shear resistant; (ii) it is of the unique class in that each molecule of mature linear DNA is like every other in the population; and (iii) its cohesive ends, which are of known structure and sequence, permit the formation of hydrogenbonded monomeric circles or higher polymers, depending upon the DNA concentration at which the annealing occurs. Furthermore, the hydrogen-bonded circles can be treated with polynucleotide ligase to form covalently closed molecules. Other advantages derive from the ability to isolate characterizable DNA fragments (either single stranded [9] or double stranded [1, 7] ) and from the extensive genetic knowledge of this bacteriophage and its hosts. Although one can readily obtain large quantities of mature linear DNA and modest quantities of the by-products listed above, it has, up to now, been fairly difficult to obtain large quantities of covalently closed molecules. Two methods that have been employed to isolate such molecules have been the in vitro sealing of hydrogen-bonded Hershey circles by polynucleotide ligase, and superinfection of an immune lysogen. The former procedure necessitates the purification of the enzyme and, in addition, results in molecules of varying degrees of superhelicity depending upon the conditions employed in the reaction mixture (3); the product, therefore, is not necessarily the same as that occurring in vivo. The yield of covalently closed circles formed in vivo by superinfection, on the other hand, is limited to a maximum of '