Jörn Glökler

Publications (28) View all

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  Available from: Jörn Glökler

  Article: Directed evolution of nucleotide-based libraries using lambda exonuclease.

  Bee Nar Lim, Yee Siew Choong, Asma Ismail, Jörn Glökler, Zoltán Konthur, Theam Soon Lim
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  ABSTRACT: Directed evolution of nucleotide libraries using recombination or mutagenesis is an important technique for customizing catalytic or biophysical traits of proteins. Conventional directed evolution methods, however, suffer from cumbersome digestion and ligation steps. Here, we describe a simple method to increase nucleotide diversity using single-stranded DNA (ssDNA) as a starting template. An initial PCR amplification using phosphorylated primers with overlapping regions followed by treatment with lambda exonuclease generates ssDNA templates that can then be annealed via the overlap regions. Double-stranded DNA (dsDNA) is then generated through extension with Klenow fragment. To demonstrate the applicability of this methodology for directed evolution of nucleotide libraries, we generated both gene shuffled and regional mutagenesis synthetic antibody libraries with titers of 2×108 and 6×107, respectively. We conclude that our method is an efficient and convenient approach to generate diversity in nucleic acid based libraries, especially recombinant antibody libraries.
  BioTechniques 12/2012; 53(6):357-64. · 2.67 Impact Factor
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  Article: Application of aptamers in therapeutics and for small-molecule detection.

  M Menger, J Glökler, M Rimmele
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  ABSTRACT: Nucleic acids that can bind with high affinity and specificity to target molecules are called "apta mers". Aptamers recognise a large variety of different molecule classes. The main focus of this chapter is small molecules as targets. Aptamers are applied complementarily to antibody technologies and can substitute antibodies or small molecules wherever their different properties, such as biochemical nature or highly discriminating capacities, are advantageous. Examples of promising applications of these versatile molecules are discussed in the field of therapeutics and biotechnology with a special view to small-molecule detection.
  Handbook of experimental pharmacology 02/2006;
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  Available from: Jörn Glökler

  Article: Next generation of protein microarray support materials: evaluation for protein and antibody microarray applications.

  Philipp Angenendt, Jim Glökler, Jens Sobek, Hans Lehrach, Dolores J Cahill
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  ABSTRACT: The performance of protein and antibody microarrays is dependent on various factors, one of which is the use of an appropriate microarray surface for the immobilisation of either protein or antibody samples. We have investigated the properties of seven new surfaces in the context of both protein and antibody microarray technology. We have demonstrated the functionality of all new slide coatings and investigated the mean signal to spotted concentration ratio, determined detection limits and calculated coefficients of variation. Moreover, new concepts for slide coatings such as dendrimer and poly(ethylene glycol)-epoxy slides were evaluated and improved qualities of novel slide surfaces were observed. Optimal slide coatings for antibody and protein chips were proposed and the requirements for both technologies were discussed.
  Journal of Chromatography 09/2003; 1009(1-2):97-104. · 4.53 Impact Factor
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  Article: A DNA aptamer with high affinity and specificity for therapeutic anthracyclines.

  Aniela Wochner, Marcus Menger, Dagmar Orgel, Birgit Cech, Martina Rimmele, Volker A Erdmann, Jörn Glökler
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  ABSTRACT: We describe the characterization of a DNA aptamer that displays high affinity and specificity for the anthracyclines daunomycin and doxorubicin, both of which are frequently used in chemotherapy. Aptamers were isolated from a pool of random sequences using a semiautomated procedure for magnetic beads. All selected aptamers displayed high affinity for the target molecule daunomycin. One aptamer was further characterized and exhibited a dissociation constant (KD) of 20 nM. To examine the aptamer's binding properties and clarify its applicability for diagnostic assays, its performance under various buffer conditions was evaluated. The aptamer proved to be very robust and not dependent on the presence of specific ions. It also tolerated a wide pH range and immobilization via 5'-biotinylation. Furthermore, a competition assay for sensitive daunomycin detection was established. This not only allows the determination of the aptamer's specificity but also allows the quantification of as little as 8.4 microg/L daunomycin and doxorubicin.
  Analytical Biochemistry 03/2008; 373(1):34-42. · 3.00 Impact Factor
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  Article: Semi-automated selection of DNA aptamers using magnetic particle handling.

  Aniela Wochner, Birgit Cech, Marcus Menger, Volker A Erdmann, Jörn Glökler
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  ABSTRACT: We have developed a semi-automatic selection procedure for DNA aptamers. Employing a robotic workstation for magnetic particle handling, this method allows for a fast, reproducible, and parallelized selection of DNA aptamers. The selection protocol is designed to provide high flexibility and versatility in terms of choice of buffers and reagents, as well as stringency of selection. Using this procedure, we have successfully isolated ligand-specific, high-affinity DNA aptamers.
  BioTechniques 10/2007; 43(3):344, 346, 348 passim. · 2.67 Impact Factor