Cloning the Pfu DNA polymerase from DNA contaminants in preparations of commercial Pfu DNA polymerase

The Pfu DNA polymerase gene was cloned from commercial Pfu DNA polymerase by semi-nested polymerase chain reaction (PCR) using a contaminant template in the commercial Pfu DNA polymerase and the activity of the Pfu DNA polymerase itself. We confirmed the occurrence of residual bacterial DNA in standard Pfu DNA polymerase preparations and demonstrated that the commercial enzyme can be used directly as a PCR template for cloning, thus eliminating the laborious and time-consuming steps of cell recovery and DNA extraction. A simple method was developed for the purification of PfuDNA polymerase by the heat-mediated (100°C, 10 min) lysis of Escherichia coli and denaturation of E. coli proteins, exploiting using the extremely thermostable properties ofPfu DNA polymerase, followed by chromatography on heparin columns. The whole purification process could be achieved within 2 h using one type of buffer, with no need for sonication or the use of toxic reagents. This advantageous alternative method is convenient and faster than previously reported methods.   Key words: Pfu DNA polymerase, DNA contamination, cloning, purification