TY - GEN A1 - Schulze, Katja A1 - Glökler, Jörn A1 - Kadolsky, Marianne A1 - Frohme, Marcus T1 - Detektion des Kartoffelspindelknollen Viroids mit Hilfe der Loop Mediated Isothermal Amplification T2 - Wissenschaftliche Beiträge 2011 N2 - PSTV ist ein hoch infektiöses Viroid, das in Kartoffeln verkleinerte und spindelähnliche Knollen verursacht. Um Ernteverlusten vorzubeugen, ist eine Detektion in frühen Infektionsstadien von großer Bedeutung. Aufgrund ihrer hohen Sensitivität und Spezifität wird die PCR als Standardnachweisverfahren für PSTV verwendet. Nachteilig an dieser Methode sind der apparative Aufwand und die zeitaufwändige Durchführung. Als viel versprechende Alternative konnte der Nachweis von PSTV mit Hilfe der Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) gezeigt werden. Hierbei handelt es sich um eine einfache und schnelle Methode, für die wenig aufwändige Laborausrüstung benötigt wird. Dabei ermöglichte eine an den Amplifikationsprozess gekoppelte Fluoreszenzreaktion die Detektion von Produkten direkt nach der Nachweisreaktion mit dem bloßen Auge (bzw. unter UV-Licht). Die Ergebnisse konnten mit Hilfe einer Real Time Detektion des auftretenden Fluoreszenzsignals bestätigt werden. N2 - PSTV is a highly infectious viroid which leads to small and spindle shaped tubers in potatoes. The detection at an early stage of infection is important to minimize loss of harvest. Normally PCR is used for the detection of PSTV, because of its high sensitivity and specificity. Disadvantages of this method are the requirement of sophisticated equipment and the time consuming process. As a promising alternative, the detection of PSTV with the reverse transcription loop mediated isothermal amplification (RT-LAMP) was shown. LAMP is a very fast and simple detection method requiring only standard laboratory equipment. A fluorescence reaction, coupled to the amplification process, allowed the detection of amplification products directly after the reaction with the naked eye (or under UV light, respectively). Real time detection of the occurring fluorescence signal was possible and confirmed the obtained results. Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus-1025 SN - 0949-8214 VL - 15 SP - 7 EP - 12 ER - TY - JOUR A1 - Endrullat, Christoph A1 - Glökler, Jörn A1 - Franke, Philipp A1 - Frohme, Marcus T1 - Standardization and quality management in next-generation sequencing JF - Applied & Translational Genomics N2 - DNA sequencing continues to evolve quickly even after > 30 years. Many new platforms suddenly appeared and former established systems have vanished in almost the same manner. Since establishment of next-generation sequencing devices, this progress gains momentum due to the continually growing demand for higher throughput, lower costs and better quality of data. In consequence of this rapid development, standardized procedures and data formats as well as comprehensive quality management considerations are still scarce. Here, we listed and summarized current standardization efforts and quality management initiatives from companies, organizations and societies in form of published studies and ongoing projects. These comprise on the one hand quality documentation issues like technical notes, accreditation checklists and guidelines for validation of sequencing workflows. On the other hand, general standard proposals and quality metrics are developed and applied to the sequencing workflow steps with the main focus on upstream processes. Finally, certain standard developments for downstream pipeline data handling, processing and storage are discussed in brief. These standardization approaches represent a first basis for continuing work in order to prospectively implement next-generation sequencing in important areas such as clinical diagnostics, where reliable results and fast processing is crucial. Additionally, these efforts will exert a decisive influence on traceability and reproducibility of sequence data. KW - next-generation sequencing KW - quality management KW - data quality KW - standardization KW - validation KW - guideline Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6637 SN - 2212-0661 VL - 10 SP - 2 EP - 9 ER - TY - JOUR A1 - Kagel, Heike A1 - Bier, Frank Fabian A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - A Novel Optical Method To Reversibly Control Enzymatic Activity Based On Photoacids JF - Scientific Reports N2 - Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report a novel optical approach to reversibly control a typical biochemical reaction by changing the pH and using acid phosphatase as a model enzyme. The reversible photoacid G-acid functions as a proton donor, changing the pH rapidly and reversibly by using high power UV LEDs as an illumination source in our experimental setup. The reaction can be tightly controlled by simply switching the light on and off and should be applicable to a wide range of other enzymatic reactions, thus enabling miniaturization and parallelization through non-invasive optical means. Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-12685 SN - 2045-2322 VL - 9 ER - TY - JOUR A1 - Kagel, Heike A1 - Jacobs, Hannes A1 - Bier, Frank Fabian A1 - Glökler, Jörn A1 - Frohme, Marcus T1 - A Novel Microtiter Plate Format High Power Open Source LED Array JF - Photonics N2 - Many photochemical or photobiological applications require the use of high power ultraviolet light sources, such as high-pressure mercury arc lamps. In addition, many photo-induced chemical, biochemical and biological applications require either a combinatorial setting or a parallel assay of multiple samples under the same environmental conditions to ensure reproducibility. To achieve this, alternative, controllable light sources, such as ultraviolet light emitting diodes (UV LEDs) with high power and spatial control are required. Preferably, LEDs are arranged in a suitable standardized 96-well microtiter plate format. We designed such an array and established the methods required for heat management and enabling stable, controllable illumination over time. KW - optics KW - photonics KW - light KW - LED array KW - High-power LED KW - 365 nm KW - microtiter plate Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-11307 SN - 2304-6732 VL - 6 IS - 1 ER - TY - JOUR A1 - Fischbach, Jens A1 - Loh, Qiuting A1 - Bier, Frank Fabian A1 - Lim, Theam Soon A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method JF - Scientific Reports N2 - We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluate inorganic pyrophosphate sensitivity in the presence of manganese as quencher and optimize conditions for an online detection. Of the selected dyes, the inexpensive alizarin red S was found to selectively detect pyrophosphate under LAMP and PCR conditions and is superior with respect to its defined red-shifted spectrum, long shelf life and low toxicity. In addition, the newly identified properties may also be useful in other enzymatic assays which do not generate nucleic acids but are based on inorganic pyrophosphate. Finally, we propose that our screening method may provide a blueprint for rapid screening of compounds for detecting inorganic pyrophosphate. KW - biochemical assays KW - fluorescent dyes KW - screening Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9248 SN - 2045-2322 VL - 7 ER - TY - JOUR A1 - Kagel, Heike A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Photoacids in biochemical applications JF - Journal of Cellular Biotechnology N2 - BACKGROUND: After excitation with light photoacids can change the pH in a solution by release of a proton. They have been used mostly for excited state proton transfer studies. In this review the general functionality and mechanisms and the subdivision of photoacids is explained. STATE OF THE ART: Different uses of photoacids are described, covering a wide range of various biochemical topics, focusing on biochemical applications. Examples for the introduced subdivisions are covered. CONCLUSIONS AND OUTLOOK: The areas in which photoacids can be employed are diverse. Photoacids have a promising future in biotechnology and biochemistry and should be considered for upcoming applications, especially in non-invasive control of biochemical reactions. Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-16123 SN - 2352-3697 VL - 4 IS - 1-2 SP - 23 EP - 30 PB - IOS Press ER - TY - JOUR A1 - Glökler, Jörn A1 - Lim, Theam Soon A1 - Ida, Jeunice A1 - Frohme, Marcus T1 - Isothermal amplifications – a comprehensive review on current methods JF - Critical Reviews in Biochemistry and Molecular Biology N2 - AbstractThe introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole. Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-15154 SP - 1 EP - 44 PB - Taylor & Francis ER - TY - JOUR A1 - Radivoievych, Aleksandar A1 - Kolp, Benjamin A1 - Grebinyk, Sergii A1 - Prylutska, Svitlana A1 - Ritter, Uwe A1 - Zolk, Oliver A1 - Glökler, Jörn A1 - Frohme, Marcus A1 - Grebinyk, Anna T1 - Silent Death by Sound: C60 Fullerene Sonodynamic Treatment of Cancer Cells JF - International Journal of Molecular Sciences N2 - The acoustic pressure waves of ultrasound (US) not only penetrate biological tissues deeper than light, but they also generate light emission, termed sonoluminescence. This promoted the idea of its use as an alternative energy source for photosensitizer excitation. Pristine C60 fullerene (C60), an excellent photosensitizer, was explored in the frame of cancer sonodynamic therapy (SDT). For that purpose, we analyzed C60 effects on human cervix carcinoma HeLa cells in combination with a low-intensity US treatment. The time-dependent accumulation of C60 in HeLa cells reached its maximum at 24 h (800 ± 66 ng/106 cells). Half of extranuclear C60 is localized within mitochondria. The efficiency of the C60 nanostructure’s sonoexcitation with 1 MHz US was tested with cell-based assays. A significant proapoptotic sonotoxic effect of C60 was found for HeLa cells. C60′s ability to induce apoptosis of carcinoma cells after sonoexcitation with US provides a promising novel approach for cancer treatment. KW - ultrasound KW - C60 fullerene KW - sonodynamic therapy KW - HeLa cells KW - apoptosis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-16877 SN - 1422-0067 VL - 24 IS - 2 PB - MDPI ER - TY - JOUR A1 - Ida, Jeunice A1 - Chan, Soo Khim A1 - Glökler, Jörn A1 - Lim, Yee Ying A1 - Choong, Yee Siew A1 - Lim, Theam Soon T1 - G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing JF - Molecules N2 - G-quadruplexes are made up of guanine-rich RNA and DNA sequences capable of forming noncanonical nucleic acid secondary structures. The base-specific sterical configuration of G-quadruplexes allows the stacked G-tetrads to bind certain planar molecules like hemin (iron (III)-protoporphyrin IX) to regulate enzymatic-like functions such as peroxidase-mimicking activity, hence the use of the term DNAzyme/RNAzyme. This ability has been widely touted as a suitable substitute to conventional enzymatic reporter systems in diagnostics. This review will provide a brief overview of the G-quadruplex architecture as well as the many forms of reporter systems ranging from absorbance to luminescence readouts in various platforms. Furthermore, some challenges and improvements that have been introduced to improve the application of G-quadruplex in diagnostics will be highlighted. As the field of diagnostics has evolved to apply different detection systems, the need for alternative reporter systems such as G-quadruplexes is also paramount. KW - G-quadruplex KW - DNAzyme KW - colorimetric KW - fluorescence KW - luminescence KW - diagnostics Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-11438 SN - 1420-3049 VL - 24 IS - 6 ER - TY - JOUR A1 - Fischbach, Jens A1 - Frohme, Marcus A1 - Glökler, Jörn T1 - Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method JF - Scientific Reports N2 - The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity. KW - DNA synthesis KW - Pathogens Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-9824 SN - 2045-2322 VL - 7 ER - TY - JOUR A1 - Devulapally, Praneeth Reddy A1 - Bürger, Jörg A1 - Mielke, Thorsten A1 - Konthur, Zoltán A1 - Lehrach, Hans A1 - Yaspo, Marie-Laure A1 - Glökler, Jörn A1 - Warnatz, Hans-Jörg T1 - Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes JF - Genome Medicine N2 - Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies. Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-10538 SN - 1756-994X VL - 10 SP - 1 EP - 12 ER - TY - JOUR A1 - Omar, Noorsharmimi A1 - Loh, Qiuting A1 - Tye, Gee Jun A1 - Choong, Yee Siew A1 - Noordin, Rahmah A1 - Glökler, Jörn A1 - Lim, Theam Soon T1 - Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer JF - Sensors N2 - G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays. KW - G-quadruplex KW - DNAzyme KW - gold nanoparticle KW - antibody Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:526-opus4-6267 SN - 1424-8220 VL - 14 IS - 1 SP - 346 EP - 355 ER -