Cloning of thermostable recombinant dna polymerase in escherichia coli / Sayeeda Zainab Hasnor

Background: The DNA polymerase from Pyrococcus furiosus (Pfu) has gained considerable attention in the field of DNA amplification as this enzyme have elevated temperature optima and have thermal stabilities that roughly correspond to the thermal extremes of the environment from which they were isolated. Due to thermostability of this enzyme, the structure and function relationships and the potential industrial applications of many thermostable enzymes such as DNA polymerases are of considerable interest to researchers. Aim: The purpose of this study is to clone the Recombinant DNA polymerase from Pyrococcus furiosus in Escherichia coli. Method: The PCR was conducted based on standard PCR protocol and was optimized under different annealing temperatures using EppendorfMastercycler Gradient to determine the optimal annealing temperature for this method. And also for further optimization, new primers batch was developed to eliminate contaminate primers. Results: The PCR based method was failed to give the result. It shows that there was no DNA amplification but only DNA ladder band can be seen. Conclusion: This study failed to amplify the Pfu DNA polymerase gene. More time needed to optimize the PCR condition.