Detektion des Kartoffelspindelknollen Viroids mit Hilfe der Loop Mediated Isothermal Amplification

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.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

Identifizierung und Charakterisierung von Metallchelat-bindenden Peptiden mittels Phage-Display

Rekombinante Proteine können verändert werden um Metallionen zu binden. Dieses Prinzip wird zur Aufreinigung mittels immobilisierter Metallchelat Chromatographie (IMAC) verwendet. Eine Fusion mit einer kurzen Sequenz von 6 Histidinresten zur Bindung von Übergangsmetallionen wie Cu2+, Ni2+, Co2+ and Zn2+ bewirkt dies. Eine Phagen-Bibliothek mit einer 15-mer Zufallssequenz wurde verwendet, um Peptide mit Affinität zu Übergangsmetallionen sowie harten Lewissäuren wie Fe3+ and Al3+ zu selektieren. Verschiedene Metallchelate sowie Stringenzen wurden bei der Selektion verwendet. Vier Selektionsrunden reichten zur Anreicherung für Metallchelat spezifischer Phagenpopulationen aus. Übergangsmetall bindende Peptide mit der größten Affinität hatten 4 Histidinreste, flankiert von hydrophoben Aminosäuren und Prolin. Die Spezifität für den Chelatbildner korrelierte mit der relativen Position des Bindungsmotifs im Peptid sowie der Hydrophobizität.Recombinant proteins can be engineered for affinity to metal ions. This principle is exploited for the purification by immobilised affinity chromatography (IMAC). Fusion of a short sequence of 6 histidine residues to a protein is sufficient for binding to transition metal ions like Cu2+, Ni2+, Co2+ and Zn2+. A phage-displayed random 15-mer library was used to select peptides binding to transition metal ions as well as to hard lewis acids such as Fe3+ and Al3+. Different formats for the immobilisation of metal ions and stringency of selection were employed. Four rounds of panning were sufficient to enrich a phage population with specific binding to the given metal chelates. Peptides binding to transition metals with highest affinity contained 4 consecutive histidine residues, flanked with hydrophobic amino acids and proline. Specificity for the chelating support material correlated with relative position of the binding motif in the sequence and hydrophobicity. As an example, a helper phage carrying a transition metal binding peptide was constructed and shown to purify by IMAC. Panning on Fe3+ yielded positively charged peptides. The best binder contained 4 lysine and two histidine residues and was shown to bind to Ni2+ in absence of imidazole. Binding to Fe3+- IDA complexes could be efficiently competed with either primary amines, phosphate or pH 8 and higher. This bispecific peptide may serve for a cascade purification on Ni2+ and Fe3+ columns, yielding higher homogeneity. Furthermore, affinity purification with iron abrogates the tedious removal of contaminating toxic heavy metal ions in traditional IMAC

A calibrated diversity assay for nucleic acid libraries using DiStRO—a Diversity Standard of Random Oligonucleotides

We have determined diversities exceeding 1012 different sequences in an annealing and melting assay using synthetic randomized oligonucleotides as a standard. For such high diversities, the annealing kinetics differ from those observed for low diversities, favouring the remelting curve after annealing as the best indicator of complexity. Direct comparisons of nucleic acid pools obtained from an aptamer selection demonstrate that even highly complex populations can be evaluated by using DiStRO, without the need of complicated calculations

Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts

Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

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

Photoacids in biochemical applications

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