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

Download

index.pdf

Date

2011

Author

Yıldız, Lütfiye

Metadata

Show full item record

Item Usage Stats

355
views

124
downloads

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.

Subject Keywords

Biotechnology., Genetics.

URI

http://etd.lib.metu.edu.tr/upload/12613099/index.pdf
https://hdl.handle.net/11511/20431

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads Öktem, Hüseyin Avni; Ozalp, V. Cengiz; Arica, M. Yakup (Wiley, 2007-01-01) A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surf...
PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus Ozturk, ZN; Ögel, Zümrüt Begüm (Springer Science and Business Media LLC, 2000-10-01) The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus p...
Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles Demir, Ayhan Sıtkı; Talpur, Farah N.; Sopaci, S. Betul; Kohring, Gert-W; ÇELİK, AYHAN (Elsevier BV, 2011-04-10) Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co2+ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and L-tagatose starting from hydroxyacetone and galactitol....
Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes Kocabıyık, Semra; Zwickl, Peter; Ozdogan, Seda (Elsevier BV, 2010-10-01) In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 205 proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylam...
RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment Hakki, EE; Akkaya, Mahinur (Elsevier BV, 2001-02-01) No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be beta alpha beta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus spe...

Citation Formats

L. Yıldız, “Isolation and characterization of Taq DNA polymerase and optimization and validation of newly designed thermal cyclers,” M.S. - Master of Science, Middle East Technical University, 2011.