Amplification and cloning of taq gene from thermus aquaticus YT-1 strain

Abstract

Introduction: DNA polymerase is the crucial enzyme in polymerase chain reaction (PCR). The polymerase derived from Thermus aquaticus was found to be superior to the historical DNA polymerase of Escherichia coli, as it can withstand the extreme denaturation temperature during the PCR procedure. This study aims to amplify and clone the Taq gene from Thermus aquaticus YT-1 strain. Methods: Thermus aquaticus YT -1 strain was cultured on Castenholz medium. The bacterial colonies were then subjected to DNA extraction and purification processes to yield DNA for the PCR. The targeted fragment of Taq gene was amplified by using Hotstart PCR technique. The isolated amplified Taq gene was then digested with restriction enzymes and ligated into pUC18 cloning vector that had also been similarly cleaved. Following that the recombinant plasmid was transformed into ampicillin resistant Escherichia coli strain DH5a. PCR screening was done to confirm the insertion of Taq DNA gene. Results: the successful isolation of the Taq polymerase gene from Thermus aquaticus YT -1 strain was confirmed by DNA sequencing of the product. The study however failed to show a significant growth of transfom1ed E. coli colonies that carried the target gene. Discussion: even though the Taq polymerase gene was successfully isolated, the failure to successfully clone it into the cloning vector is speculated to be related to the relatively large size of the Taq gene that requires further optimization procedures to be perfonned on the employed methodology. Conclusion: the isolated Taq polymerase gene could be used as a foundation material for the development of further optimized and/or improvised gene cloning and expression techniques in a near future subsequent study.