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DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

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KRANASTER, Ramon, 2010. DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

@phdthesis{Kranaster2010polym-9558, title={DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution}, year={2010}, author={Kranaster, Ramon}, address={Konstanz}, school={Universität Konstanz} }

Kranaster, Ramon 2011-03-24T18:12:44Z application/pdf In this PhD thesis, several projects about the functional analysis and recruitment of mutated DNA polymerases for improved biotechnological applications were investigated. Discrimination of incorrect pairing single nucleotides is of fundamental importance for the enzyme-aided detection of single nucleotide variations (single nucleotide polymorphisms (SNPs)). It could be demonstrated that both chemically modified primer probes which are thiolated at the 2-position of thymidine as well as mutated DNA polymerases were able to increase single-nucleotide discrimination.<br />Based on these findings a DNA chip based system for the multiplex detection of single nucleotide polymorphisms (SNPs) was established. For that purpose, a mutated DNA polymerase from Pyrococcus furiosus with improved single nucleotide discrimination properties is used for selective microarrayed primer extensions. It is shown that the mutated DNA polymerase in combination with unmodified primer strands fulfils the demands on solid support and obviates the need for chemical modifications of the primer probes as required before. The system depicted herein could provide the basis for further advancements in microarrayed nucleic acid diagnostics using tailor-made enzymes.<br />Until now, all reported methods for DNA polymerase evolution are restricted to a single enzyme property, for example, increased selectivity or the ability to efficiently process DNA lesions. Thus, a new microarrayed device was developed to overcome these obvious limitations that allow the multiplexed screening of several enzyme features in parallel:<br />The approach is based on the spatial separation of different covalently attached DNA substrates on a glass slide and their selective addressing by oligonucleotide hybridization. This system, termed oligonucleotide-addressing enzyme assay (OAEA), enables multiplexed simultaneous profiling of DNA polymerases in nanoliter volumes in terms of their different properties. OAEA can be used for the simultaneous and multiplexed profiling of several enzyme features with high throughput. Additionally, other DNA-modifying enzymes like ligases and endonucleases can be included in multiplex directed evolution approaches using OAEA. As a first successful demonstration it was used to identify enzymes with altered properties out of a library of DNA polymerase mutants.<br />A functional chimeric DNA polymerase could be obtained by fusion of a wild-type 5 ́- 3 ́nuclease domain with a recently described N-terminally shortened DNA polymerase from Thermus Aquaticus, which exhibits a significantly increased reverse transcription activity. The new enzyme (named as Taq M1) was created to improve RNA pathogen detection systems for pathogens like Dobrava viruses. It could be demonstrated that the fusion of polymerase- and 3 ́nuclease-domain to constitute Taq M1 has no effect on the originally polymerase- and nuclease function and activities. Additionally, Taq M1 was used in applied TaqMan RNA detection assays: Without optimisation of reaction conditions Taq M1 provided detection sensitivities compared to commercially available one-step RT PCR systems, which are based on enzyme blends. Taq M1 is highly recommended for the use of one-step RT PCR, especially if high transcription temperatures are desired to melt stable secondary structures of RNA targets.<br />In my last project, functional studies were conducted with the N-terminally shortened DNA polymerase from Thermus Aquaticus (KlenTaq). Recently obtained crystal structures of KlenTaq in complex with both an abasic site harbouring template and a blunt-ended primer template substrate (Schnur et al. and personal communication with S.Obeid), revealed that amino acid tyrosine 671 plays an important role in the template-less selection of the incorporated nucleotide. Tyrosine 671 thereby mimics the steric constraints of a pyrimidine template base resulting in the favoured incorporation of purine bases (A and G). Mutation of tyrosine into alanine (Y671A) results in a dramatic drop of catalytic activity. Mutation of the aromatic tyrosine into the also aromatic but steric more demanding tryptophane results in the favoured incorporation of pyrimidine bases (T and C). These findings could be proved by single nucleotide incorporation studies and enzyme kinetic measurements. deposit-license 2011-03-24T18:12:44Z deu Kranaster, Ramon DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution 2010

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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