30 research outputs found
DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution
No full textIn 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.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.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: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.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.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.publishe
A mutated thermostable Thermus aquaticus DNA polymerase with reverse transcriptase activity for one step RNA pathogen detection
No full textInternational audienceWe describe the cloning and characterisation of a mutated thermostable DNA polymerase from Thermus aquaticus (Taq) which exhibits an increased reverse transcriptase activity and is therefore designated for one step PCR pathogen detection using established real-time detection methods. We demonstrate that this Taq polymerase mutant (Taq M1) has similar PCR sensitivity and nuclease activity as the respective Taq wild-type DNA polymerase. In addition and in marked contrast to the wild-type, Taq M1 exhibits a significantly increased reverse transcriptase activity especially at high temperatures (> 60°C). RNA generally hosts highly stable secondary structure motifs such as hairpins and G-quadruplexes which complicate or in the worst case obviate reverse transcription (RT). Thus, RT at high temperatures is desired to weaken or melt secondary structure motifs. To demonstrate the ability of Taq M1 for RNA detection of pathogens we performed TaqMan probe-based diagnostics of Dobrava viruses by one step RT-PCR. Indeed, we found comparable detection sensitivities compared to commercial available RT-PCR systems without further optimization of reaction parameters thus making this enzyme highly suitable for any PCR probe based RNA detection method
Fingerabdrücke von DNA-Polymerasen : mehrfache simultane Enzym-Charakterisierung auf DNA-Arrays
No full textDNA-Polymerasen kommen bei einer ganzen Fülle von biotechnologischen Anwendungen zum Einsatz, insbesondere bei der Polymerasekettenreaktion (PCR), genetischen Klonierungen, Genomsequenzierungen und bei diagnostischen Anwendungen.[1] Für Klonierungen sind hoch prozessive und möglichst fehlerfreie DNA-Polymerasen erwünscht, da diese zu kürzeren Verlängerungszeiten und zu einer robusteren Amplifikation mit großer Ausbeute führen. Eine höhere Genauigkeit von DNA-Polymerasen könnte Genomsequenzierungen und diagnostische Anwendungen verlässlicher machen.[2] Die Amplifikation von prähistorischen DNAProben erfordert DNA-Polymerasen mit einem erweiterten Substratspektrum, damit typische DNA-Schäden effizient überlesen werden können.[3] Um die Effizienz von forensischen DNA-Tests zu erhöhen, braucht man DNA-Polymerasen, die resistent gegen Inhibitoren aus Blut- und Erdproben sind und somit eine PCR ohne vorherige DNA-Reinigung möglich machen.[4] Weitere Verbesserungen von DNA-Polymerasen sind z.B. notwendig, um den Anforderungen von DNA-Sequenzierungen einzelner Moleküle zu genügen, die auf dem effizienten Einbau modifizierter Nucleotide beruhen.[5] Insgesamt werden dringend maßgeschneiderte, künstliche DNA-Polymerasen benötigt, die zu robusteren und spezifischeren Reaktionssystemen führen.publishe
Engineered DNA Polymerases in Biotechnology
No full textDNA polymerases are the enzymes that catalyse all DNA synthesis in Nature often with astounding speed and accuracy. Consequently, their features as molecular machines are exploited in a wide range of biotechnological applications. Some features are highlighted in the following. For example, DNA polymerases are useful enzymes to detect genomic alterations that can lead to the development of certain diseases such as cancer or to promote toxic side effects of drugs. Methods for the detection of single-nucleotide polymorphisms, copy-number variations and somatic copy-number alterations are important for the realisation of personalised medicine.[1 3] Additionally, new DNA sequencing technologies aim to achieve the $ 1000 genome that might further drive a new era of specific pharmaceutical treatments and diseaseprevention strategies based on an individual s genome.[4 7] In many DNA diagnostic and sequencing methods, the accurate action of a DNA polymerase to incorporate the right nucleotide with high selectivity, according to the Watson Crick rule, is crucial.publishe
Taking Fingerprints of DNA Polymerases : Multiplex Enzyme Profiling on DNA Arrays
No full textDNA polymerases are used in a plethora of biotechnical applications, especially in the polymerase chain reaction (PCR), genetic cloning procedures, genome sequencing, and diagnostic methods.[1] Highly processive and accurate DNA polymerases are desired for cloning procedures in order to give shorter extension times as well as more robust and highyield amplification. A higher DNA polymerase fidelity may increase the reliability of genome sequencing and diagnostic systems.[2] Amplification of ancient DNA samples requires DNA polymerases with an increased substrate spectrum to efficiently overcome typical DNA lesions.[3] To enhance the efficiency of forensic DNA testing, DNA polymerases resistant to inhibitors from blood and soil allow PCR without prior DNA purification.[4] Further improvements of DNA polymerases are required, for example, to meet the requirements of real-time DNA single-molecule sequencing, which relies on the ability ofDNApolymerases to efficiently process modified nucleotides.[5] Overall, customized and artificially engineered DNA polymerases that lead to more robust and specific reaction systems are urgently needed.publishe
New Strategies for DNA Polymerase Library Screening
No full textEngineered enzymes are of increasing importance for a plethora of biotechnical applications. Especially DNA polymerases are workhorses in biochemical technologies in particular the polymerase chain reaction (PCR), cDNA cloning procedures, genome sequencing and in diagnostic applications. DNA polymerase mutant libraries can be used for the screening of non-standard reaction conditions or substrates e.g. the efficient amplification of difficult templates like ancient DNA. We are convinced that these fascinating enzymes can be optimized and costum-made for a specific application to result in more robust and reliable systems. To our knowledge, all known screening methods for DNA polymerase mutants are focused and thus limited to the screening of a single reaction or one new function. We developed improved strategies for multiplexed DNA polymerase screening that will be presented.publishe
Improved LNA probe-based assay for the detection of African and South American yellow fever virus strains
No full textBackground: Real-time assays for Yellow fever virus (YFV) would help to improve acute diagnostics in outbreak investigations. Objectives: To develop a real-time assay for YFV able to detect African and South American strains. Study design:Three short probe (14-18 nt) formats were compared and a plasmid-transcribed RNA standard was used to test the performance of the assays. Additionally the new TaqM1 enzyme was tested. Results: A locked nucleotide probe (LNA probe) performed best with an analytical sensitivity of 10 RNA molecules detected. 44 African and 10 South American strains were detectable. One South American strain from 1984 had a one-nucleotide deviation in the hybridisation sequence for which the LNA probe had to be adapted. Comparison of enzymes revealed that not all enzymes are suitable for LNA probes. Conclusion: The developed LNA probe based YFV real-time PCR performed best in an enzyme mix and less efficient using multifunctional enzymes
Hydrophobic Amino Acid and Single-Atom Substitutions Increase DNA Polymerase Selectivity
No full textSummaryDNA polymerase fidelity is of immense biological importance due to the fundamental requirement for accurate DNA synthesis in both replicative and repair processes. Subtle hydrogen-bonding networks between DNA polymerases and their primer/template substrates are believed to have impact on DNA polymerase selectivity. We show that deleting defined interactions of that kind by rationally designed hydrophobic substitution mutations can result in a more selective enzyme. Furthermore, a single-atom replacement within the DNA substrate through chemical modification, which leads to an altered acceptor potential and steric demand of the DNA substrate, further increased the selectivity of the developed systems. Accordingly, this study about the impact of hydrophobic alterations on DNA polymerase selectivity—enzyme and substrate wise—further highlights the relevance of shape complementary and polar interactions on DNA polymerase selectivity
Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries
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