A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid

A general method has been developed for the deletion of restriction endonuclease sites in bacterial plasmid DNA. The procedure involves partial digestion of the covalently closed circular plasmid DNA with an appropriate restriction endonuclease under conditions which allow accumulation of unit-length linear DNA molecules, a controlled digestion of the exposed 5' ends with the lambda 5'-exonuclease, and in vivo recircularization of the resulting linear DNA in a bacterial host cell. The method has been used for the deletion of one of the two EcoRI sites in the plasmid pML2 (colE1-Km). Two of the resulting plasmids, pCR1 and pCR11, have a single EcoRI cleavage site, but retain genetic determinants specifying resistance to colicin E1 and kanamycin, and thus may be useful as vectors for the cloning and amplification of DNA in bacteria.