Cloning of human gamma glutamyl transferase (GGT) gene / Izzati Mohd Farok

The y-glutamyl transferase (GGT) plays an important role in the process of catalyzing the degradation of glutathione. GGT is widely used as a marker in preneoplastic lesions in the liver during chemical carcinogenesis. In this study, primers were designed without the restriction sites to investigate the success of cloning. The aim of this study was to clone GGT gene in plasmid by means of TA cloning. Methods used in this study were purification of human liver cDNA, PCR for gene amplification, TOPO TA cloning, transformation into E.coli and DNA sequencing. PCR was conducted under different annealing temperatures using Takara PCR Thermal Cycler Dice™ Gradient model to determine the optimal annealing temperature. TOPO TA cloning process with high cloning efficiency was completed in 5 minutes. Transformation was done using the One Shot® Chemical Competent E.coli. DNA sequencing was also performed using the plasmid samples from transformation process to validate the insert. Plasmid extraction yielded 168 ng/µl of liver cDNA. The PCR based method obtained an optimal annealing temperature for GGT amplification, which was at 47°C. Transformation was a success by using E.coli as a host where a 10 ml of LB broth was used and incubated at 3 7°C for 16 hours. Cloning of the GGT gene is important as it opens ways for further studies to look into its association with cancer and help in development of medication that prevents the progression of tumors. There is no specific treatment for patients with GGT deficiency and thus, researchers may develop protein-based drugs for them. GGT cloning can help in the establishment of gene collection in DNA libraries.