Polishing with T4 or Pfu polymerase increases the efficiency of cloning of PCR fragments.

Several of the polymerases widely used for PCR, such as Taq DNA polymerase, have been found to exhibit terminal deoxynucleotide transferase activity (1, 2, 3). This terminal transferase activity, for the most part is limited to the addition of a single nucleotide and has been named 'extendase' activity. In a widely cited study, Clark has shown that when the 3' base is a cytosine nucleotide, the Taq DNA polymerase adds predominately a single A base. This result forms the basis for the T/A cloning procedure. Although Clark's results were true for 3' C-ending fragments, Hu (3) has shown that the extended nucleotide is dependent on the specific nucleotide present at the 3' end of the synthetic double-stranded polydeoxyoligonucleotide. Different polymerases, such as Taq, T7, Klenow and Vent have different extendase characteristics with regards to which base is added at the 3' DNA ends. The polymerases derived from T4 bacteriophage and the hyperthermophilic archea Pyrococcus furiosus (available from Stratagene, La Jolla, CA) do not contain any extendase activity and in all cases were found to leave bluntended molecules.