In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome

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Abstract
Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase large fragment as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, [HpaII, EcoRI and HindIII] restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected [rat nephroma] cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.