No description available.

Oligonucleotides enable many biotechnological applications; however they are easily degraded by nucleases. Many nucleotides modified at the 2’ position are degraded at decreased rates which improves oligonucleotide utility. Most applications of oligonucleotides rely on enzymatic synthesis. Unfortunately, native DNA polymerases do not recognize most useful modified nucleotide substrates. Directed evolution has been used to identify mutants of Taq DNA polymerase I (Taq) that recognize substrates with 2’ modifications. While mutant enzymes capable of modified nucleotide addition have been identified, to date, all of these enzymes are limited by their inability to synthesize full length modified DNA. Despite considerable efforts to evolve new activity there has been little work done to quantitatively characterize these evolved enzymes. This thesis work presents efforts to synthesize modified primers that will help comparatively and quantitatively characterize three enzymes previously evolved to recognize 2’ modified substrates. Using the methods developed in this thesis project, our lab will be able to characterize the relationship between the number of modified nucleotides in the primer terminus and the rate of modified and unmodified nucleotide addition. Future work will identify key enzymatic steps that limit extension in these enzymes with implications for the future design of Taq mutants capable of synthesizing long 2’ modified oligonucleotides.

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&reg / 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.