Isolation and characterization of taq DNA polymerase and optimization and validation of newly designed thermal cyclers

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2011

Öğrenci: LÜTFİYE YILDIZ

Danışman: HÜSEYİN AVNİ ÖKTEM

Özet:

Amplification of target DNA in vitro via polymerase chain reaction (PCR) is a widely used scientific technique in molecular biology. This method relies on repeated heating and cooling cycles of the DNA and enzyme mixture, resulting with the enzymatic replication of the DNA. A heat stable Taq DNA polymerase and a thermal cycler that enables repeated heating/cooling cycles are the two key components of the PCR. In this study we have produced a high activity Taq DNA polymerase and used this enzyme to validate and optimize two newly developed thermal cyclers- a conventional and a capillary thermal cycler. Taq DNA polymerase gene was amplified from Thermus aquaticus DNA, was cloned and overexpressed using Gateway® recombination cloning technology. Highly active Taq DNA polymerase enzyme was purified from E.coli and its activity was tested by PCR, using different sources of DNA. Our results showed that the enzyme activity of the produced Taq DNA polymerase was not significantly different from the commercial available Taq DNA polymerase. To further characterize the purified enzyme, endonuclease and nicking activities were also tested to be absent. The fidelity of the purified Taq DNA polymerase was also tested and found to be the same as the commercially available Taq polymerases. In this study, in addition to the production of a Taq polymerase, optimization studies for two new thermal cyclers, a conventional and a capillary, was also carried out. The conventional thermal cycler was found to be as efficient as the commercially available thermal cyclers in the 95% confidence interval. The capillary thermal cycler was tested as a proof of concept and our results showed that it works less efficiently due to the insufficient insulation and capillary tubes being longer than the capillary tube holder.