In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites.

Abstract

A method for mutagenizing circular DNA molecules was developed that uses synthetic oligodeoxynucleotide restriction sites as mutagens. A single synthetic restriction site is introduced at random by cleaving circular DNA with a nonspecific double-strand endonuclease. The restriction site is then ligated to the ends and the molecule is subsequently recircularized. These small additions to the genome are mapped by digestion with the appropriate [EcoRI, BamHI, HincII and HaeII] restriction enzyme. Rear-rangements such as duplications and deletions can be engineered at will by using the added restriction sites. This technique was used to produce a fine-structure map of RSF1050, a ColE1 derivative [from Escherichia coli], 60% of which is a transposable DNA sequence encoding the TEM .beta.-lactamase (Tn3). A subset of the mutations, mapping within a narrow region of Tn3, result in an increased frequency of Tn3 transposition; mutations in other regions abolish transposition entirely.