@misc{SchulzeGloeklerKadolskyetal.2011, author = {Schulze, Katja and Gl{\"o}kler, J{\"o}rn and Kadolsky, Marianne and Frohme, Marcus}, title = {Detektion des Kartoffelspindelknollen Viroids mit Hilfe der Loop Mediated Isothermal Amplification}, series = {Wissenschaftliche Beitr{\"a}ge 2011}, volume = {15}, journal = {Wissenschaftliche Beitr{\"a}ge 2011}, issn = {0949-8214}, doi = {10.15771/0949-8214_2011_1_1}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus-1025}, pages = {7 -- 12}, year = {2011}, abstract = {PSTV ist ein hoch infekti{\"o}ses Viroid, das in Kartoffeln verkleinerte und spindel{\"a}hnliche Knollen verursacht. Um Ernteverlusten vorzubeugen, ist eine Detektion in fr{\"u}hen Infektionsstadien von großer Bedeutung. Aufgrund ihrer hohen Sensitivit{\"a}t und Spezifit{\"a}t wird die PCR als Standardnachweisverfahren f{\"u}r PSTV verwendet. Nachteilig an dieser Methode sind der apparative Aufwand und die zeitaufw{\"a}ndige Durchf{\"u}hrung. 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, f{\"u}r die wenig aufw{\"a}ndige Laborausr{\"u}stung ben{\"o}tigt wird. Dabei erm{\"o}glichte 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 best{\"a}tigt werden.}, language = {de} } @article{EndrullatGloeklerFrankeetal.2016, author = {Endrullat, Christoph and Gl{\"o}kler, J{\"o}rn and Franke, Philipp and Frohme, Marcus}, title = {Standardization and quality management in next-generation sequencing}, series = {Applied \& Translational Genomics}, volume = {10}, journal = {Applied \& Translational Genomics}, issn = {2212-0661}, doi = {10.1016/j.atg.2016.06.001}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-6637}, pages = {2 -- 9}, year = {2016}, abstract = {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.}, language = {en} } @article{OmarLohTyeetal.2013, author = {Omar, Noorsharmimi and Loh, Qiuting and Tye, Gee Jun and Choong, Yee Siew and Noordin, Rahmah and Gl{\"o}kler, J{\"o}rn and Lim, Theam Soon}, title = {Development of an Antigen-DNAzyme Based Probe for a Direct Antibody-Antigen Assay Using the Intrinsic DNAzyme Activity of a Daunomycin Aptamer}, series = {Sensors}, volume = {14}, journal = {Sensors}, number = {1}, issn = {1424-8220}, doi = {10.3390/s140100346}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-6267}, pages = {346 -- 355}, year = {2013}, abstract = {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.}, language = {en} } @article{GloeklerLimIdaetal.2021, author = {Gl{\"o}kler, J{\"o}rn and Lim, Theam Soon and Ida, Jeunice and Frohme, Marcus}, title = {Isothermal amplifications - a comprehensive review on current methods}, series = {Critical Reviews in Biochemistry and Molecular Biology}, journal = {Critical Reviews in Biochemistry and Molecular Biology}, publisher = {Taylor \& Francis}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-15154}, pages = {1 -- 44}, year = {2021}, abstract = {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.}, language = {en} } @article{RadivoievychKolpGrebinyketal.2023, author = {Radivoievych, Aleksandar and Kolp, Benjamin and Grebinyk, Sergii and Prylutska, Svitlana and Ritter, Uwe and Zolk, Oliver and Gl{\"o}kler, J{\"o}rn and Frohme, Marcus and Grebinyk, Anna}, title = {Silent Death by Sound: C60 Fullerene Sonodynamic Treatment of Cancer Cells}, series = {International Journal of Molecular Sciences}, volume = {24}, journal = {International Journal of Molecular Sciences}, number = {2}, publisher = {MDPI}, issn = {1422-0067}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-16877}, year = {2023}, abstract = {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.}, language = {en} } @article{IdaChanGloekleretal.2019, author = {Ida, Jeunice and Chan, Soo Khim and Gl{\"o}kler, J{\"o}rn and Lim, Yee Ying and Choong, Yee Siew and Lim, Theam Soon}, title = {G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing}, series = {Molecules}, volume = {24}, journal = {Molecules}, number = {6}, issn = {1420-3049}, doi = {10.3390/molecules24061079}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-11438}, year = {2019}, abstract = {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.}, language = {en} } @article{FischbachFrohmeGloekler2017, author = {Fischbach, Jens and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) - A New Versatile Isothermal Amplification Method}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/s41598-017-08067-x}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-9824}, year = {2017}, abstract = {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.}, language = {en} } @article{DevulapallyBuergerMielkeetal.2018, author = {Devulapally, Praneeth Reddy and B{\"u}rger, J{\"o}rg and Mielke, Thorsten and Konthur, Zolt{\´a}n and Lehrach, Hans and Yaspo, Marie-Laure and Gl{\"o}kler, J{\"o}rn and Warnatz, Hans-J{\"o}rg}, title = {Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes}, series = {Genome Medicine}, volume = {10}, journal = {Genome Medicine}, issn = {1756-994X}, doi = {10.1186/s13073-018-0542-5}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-10538}, pages = {1 -- 12}, year = {2018}, abstract = {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.}, language = {en} } @article{KagelFrohmeGloekler2018, author = {Kagel, Heike and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Photoacids in biochemical applications}, series = {Journal of Cellular Biotechnology}, volume = {4}, journal = {Journal of Cellular Biotechnology}, number = {1-2}, publisher = {IOS Press}, issn = {2352-3697}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-16123}, pages = {23 -- 30}, year = {2018}, abstract = {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.}, language = {en} } @article{KagelBierFrohmeetal.2019, author = {Kagel, Heike and Bier, Frank Fabian and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {A Novel Optical Method To Reversibly Control Enzymatic Activity Based On Photoacids}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/s41598-019-50867-w}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-12685}, year = {2019}, abstract = {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.}, language = {en} } @article{KagelJacobsBieretal.2019, author = {Kagel, Heike and Jacobs, Hannes and Bier, Frank Fabian and Gl{\"o}kler, J{\"o}rn and Frohme, Marcus}, title = {A Novel Microtiter Plate Format High Power Open Source LED Array}, series = {Photonics}, volume = {6}, journal = {Photonics}, number = {1}, issn = {2304-6732}, doi = {10.3390/photonics6010017}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-11307}, year = {2019}, abstract = {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.}, language = {en} } @article{FischbachLohBieretal.2017, author = {Fischbach, Jens and Loh, Qiuting and Bier, Frank Fabian and Lim, Theam Soon and Frohme, Marcus and Gl{\"o}kler, J{\"o}rn}, title = {Alizarin Red S for Online Pyrophosphate Detection Identified by a Rapid Screening Method}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/srep45085}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:526-opus4-9248}, year = {2017}, abstract = {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.}, language = {en} }