Production and Evaluation of Taq DNA Polymerase
CHANVIT LEELAYUWAT, Ph.D.*,**, RATANAPORN PAECHAIYAPHUM***, ARUNRA T ROMPHRUK, M.Sc. *, **, THIDA SRISUK***, TEMDUANG LIMPAIBOON, Ph.D.****, AMORNRAT ROMPHRUK, M.Sc.**, *****
Affiliation : * Department of Clinical Immunology, Faculty of Associated Medical Sciences, ** CMII-KKU Institutional Cooperative Centre (CKICC), Faculty of Associated Medical Sciences, *** Fourth year students, Faculty of Associated Medical Sciences, **** Department of Clinical Chemistry, Faculty of Associated Medical Sciences, ***** The Central Blood Transfusion Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in com- parison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protien with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.
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