Amplification of human UGT1A1 using PCR for use in cloning and expression / Ahmad Shafie

UDP-glucuronosyltransferase (UGT1A1) is one of the families of enzymes which catalyze the transfer of glucuronic acid to a range of endogenous compounds and xenobiotic during Phase II drug metabolism. It facilitates the elimination of compounds in either urine or bile. The cloning of this enzyme in vitro thus allows further studies including drug-drug, drug-herb interaction studies to be performed. In this project, we aim to amplify UGT1A1 gene from human liver DNA bank which can be used for subsequent analysis. Specific primers flanking the complete UGT1A1 coding region was designed. The specificity of the primers were evaluated using Oligo Explorer 1.2 software. A specific PCR protocol was developed to amplify the gene from the human liver DNA which was used as the template. The amplicon targeted composes of 1602 base pairs of nucleotides (533 amino acids). The gene was amplified using PCR protocol that was optimized in house. Gel electrophoresis was performed to confirm the success of amplification. The band of interest which was 1602 base pairs in size was observed under UV transilluminator after gel electrophoresis. UGT1A1 was successfully amplified from human liver DNA using PCR. The amplicon can then be used for cloning and expression of UGT1A1 enzyme.