Figure 1. Means to clone single segments into a plasmid in vitro.

A. TA cloning. A PCR product is generated with Taq polymerase, which adds a non-template-derived 3′ deoxyadenosine to DNA ends. TA cloning vectors are supplied by the manufacturer and have a 3′ deoxythymidine added to their ends by TdT. Annealing of the TA overhangs allows for efficient ligation by T4 DNA ligase. The resulting plasmid carries the PCR segment flanked by the additional A/T residues. Note that blunt-ended PCR products generated by proofreading enzymes can also be cloned by this method after a brief incubation with Taq polymerase

B. Topoisomerase cloning. Vaccinia virus DNA Topoisomerase I can cleave and rejoin ds DNA. Topo cloning vectors are supplied by the manufacturer with Topoisomerase I covalently linked to the linearized vector, allowing ligase free cloning of PCR products.