Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases.

A method for creating a group of deletion subclones for DNA sequencing by partial digestion of M13 bacteriophage constructions is outlined. The M13 construct is linearized at a unique site and then subjected to partial digestion with a frequent-cutting restriction endonuclease. The insert is truncated at different locations. The vector DNA is also partially digested. The products of a single partial digestion are repaired, recircularized by ligation, and used for bacterial transfection to generate subclones with a spectrum of deletions in the insert; most deletions in the vector DNA will disrupt vital viral genes and will thus disappear in the transfection. The subclones are sorted by size by gel electrophoresis of single-stranded viral DNA. This method is simpler and thus may be more reliable than established subcloning schemes.