Ph.D. Theses
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Browsing Ph.D. Theses by Author "Bilgin, Neşe."
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Item Characterization of thermophilic gene expression enzymes(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çağlayan, Melike.; Bilgin, Neşe.The DNA polymerase I genes of seven newly identified Geobacillus species within the family Bacillaceae, were cloned, sequenced and overexpressed in Escherichia coli. The polA gene of these species encodes DNA polymerase I of 878 amino acid residue protein with a predicted molecular weight of 99.3 kDa. Similarity analyses suggested that DNA polymerases belong to family A polymerases and lack 3'-5' exonuclease activity. The complete coding sequences of the genes were submitted into the GenBank. The recombinant (His)6-tagged DNA polymerases were purified by Ni2+-affinity chromatography and the homogeneous proteins were obtained after TEV protease digestion. DNA polymerases from Geobacillus anatolicus (Gana DNApolI) and Geobacillus kaue strain NB (Gkaue DNApolI) were further characterized in vitro and optimum conditions with respect to temperature, pH, monovalent and divalent ions were determined. Geobacillus DNA polymerase I fragment (GF DNApolI) was cloned and purified after homology modeling using Bacillus DNA polymerase I fragment (BF) as the model protein structure. The accuracy of GF DNApolI was measured by two M13 based fidelity assays which score errors produced during in vitro DNA synthesis of the lacZα complementation gene in M13mp2 DNA at 37°C, 50°C and 72°C. Base substitution errors increase three-fold when temperature is raised from 37°C to 72°C. DNA sequencing of the phage mutants showed that some of the base substitutions are more temperature sensitive than others. The most common base substitution error is the misincorporation of dGMP opposite to template G. Single nucleotide incorporations for both correct and for incorrect nucleotides were also studied under single-turnover conditions at 22°C, 37°C and 50°C. For both correct and incorrect dNTP insertions, the rate of polymerization, kpol, increased (seven- and four-fold, respectively) when temperature is raised from 22°C to 50°C, whereas only a slight change in Kd was observed. As a result, kinetic efficiency of the enzyme (kpol/Kd) shows five-fold increase over this temperature range.Item Development of single cell genetic testing strategies : assessment of MHC compatibility, meiotic recombinations and beta-thalassemia in human embryos(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Taylan, Fulya.; Altıok, Ender.; Bilgin, Neşe.Single cell molecular biology, a relatively new scientific branch, is promising to study unique questions and leading to novel applications in biology and medicine. Single cell studies have been challenged by difficulties in selection and isolation of appropriate cells, low amplification efficiencies, allele drop outs, PCR contaminations and inefficiency of conventional analysis strategies. This study has explored the possibilities of analyzing multiple genetic conditions particularly concerning the beta globin and the HLA regions in human embryos. The HLA genes, beta-globin gene, and the associated microsatellites have been amplified simultaneously by multiplex PCR. DNA sequencing has been optimized for high resolution genotyping. The real-time PCR and melting curve analysis have been adapted for the first time for rapid and reliable analysis of the HLA compatibility. Use of microsatellites of the extended HLA locus has enabled more accurate and efficient detection of the allele drop outs, contaminations and recombinations. Amplification and informative detection have been obtained for 1012 blastomeres out of 1180 human embryos used in this study, giving a detection rate of 86%. A total of 122 (13%) embryos were found unaffected from beta thalassemia and had identical genotype at ten HLA regions. Transfer of 94 embryos that have resulted into 16 pregnancies with 14 healthy offsprings indicates the feasibility of the single cell applications for preventive medical approaches. Microsatellite typing of the extended HLA locus has enabled to study the characteristics of the meiotic recombinations in human embryos. The recombination rate was determined as 0.44 cM/Mb, 2.1 fold less compared to the general genomic recombination rate of 0.92 cM/Mb. It was 3.83 fold higher in the maternal MHC regions compared to the paternal MHC regions. Upto 14 fold difference was observed among individuals. Breakpoints of recombinations in the class II region were clustered between the DRB1 and DPB1 genes covering the TAP1 and TAP2 genes.