Molecular cloning of DNA fragments produced by restriction endonucleases Sa1I and BamI.

Abstract

The highly specific restriction endonucleases SalI and BamI produce DNA fragments with complementary, cohesive termini that can be covalently joined by DNA ligase. The Escherichia coli kanamycin resistance factor pML21 has 1 SalI site, at which DNA can be inserted without interfering with the expression of drug resistance or replication of the plasmid. A more convenient cloning vehicle can be made with the tetracycline resistance factor pSC101, since insertion of DNA either at its single site for SalI or at that for BamI inactivates plasmid-specified drug resistance but not replication. To take advantage of this insertional inactivation, pSC101 was joined to a ColE1-ampicillin resistance plasmid having no SalI site, and to a ColE1-kanamycin resistance plasmid having no BamI site. Chimeras formed with the resulting hybrid vehicles can be identified simply by replica plating. These 3 vehicles, which all replicate under relaxed control, were used to clone and amplify Drosophila melanogaster DNA fragments.