Specific φX174 DNA Fragments as Internal Markers for the Mapping of φX174 DNA Molecules in the Electron Microscope

Abstract

φX174 RF II^‡ molecules were constructed in vitro by annealing circular φX174 viral DNA and linear φX174 complementary strand DNA. When a specific fragment of φX174 RF purified from a digestion with the restriction endonuclease Hind from Hemophilus influenzae was included in the renaturation mixture φX174 RF II molecules containing a loop corresponding in size to the fragment were seen. This was confirmed for three different DNA fragments. φX174 RF II molecules were constructed in the presence of two restriction fragments. In addition to molecules containing one loop corresponding in size to one of the fragments, molecules containing two loops were seen. The two loops corresponded in size to the two restriction fragments and the distances between the two loops corresponded to the distances between the two fragments on the Hind cleavage map of φX174 RF. By including two fragments of distinguishable size that map asymmetrically on the cleavage map in the renaturation mixture, φX174 RF II molecules were constructed which could be oriented on the cleavage map. Using the two loops as internal markers the φX174 genetic map was superimposed on the electron micrographs of these molecules.