Cloning of the human thiopurine methyltransferase (TMPT) gene fragment for generation of copy numbers in absolute real time PCR / Ahmad Firdaus Mohd Azhar

Thiopurine Methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs. The enzyme has been found to be polymorphic resulting in wide phannacokinetic and pharmacodynamic differences among patients. Outcome of therapy has been correlated with the expression of protein in different patients. A quantitative PCR method which measures the expression of gene is thus useful in clinical setting. Patients prescribed thiopurine should be monitored with respect to the protein expression for appropriate dosing of drugs. The aim of the study is to clone the TP MI' gene for use in quantitative PCR process. The clone would be used for the generation of calibration curve to allow an absolute quantitative PCR method to be developed. Specific amplification of the gene of interest was performed by specific primer designed. Subsequently, the DNA encoding for the correct gene was transformed into E-co/i. Specific primer was designed and the gene of interest was successfully amplified. The bacterial are successfully grown in the agar plate but the screening process does not show positive result in the colony selected.