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

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