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Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers.
Strzelecki, Patryk; Shpiruk, Anastasiia; Cech, Grzegorz M; Kloska, Anna; Hébraud, Pascal; Beyer, Nicolas; Busi, Florent; Grange, Wilfried.
Afiliación
  • Strzelecki P; Université de Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504, F-6700, Strasbourg, France; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
  • Shpiruk A; Université de Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504, F-6700, Strasbourg, France.
  • Cech GM; Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
  • Kloska A; Department of Medical Biology and Genetics, Faculty of Biology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland.
  • Hébraud P; Université de Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504, F-6700, Strasbourg, France.
  • Beyer N; Université de Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504, F-6700, Strasbourg, France.
  • Busi F; Université Paris Cité, CNRS, Unité de Biologie Fonctionnelle et Adaptative, UMR 8251, F-75013, Paris, France; Université Paris Cité, UFR Sciences du vivant, F-75013, Paris, France. Electronic address: florent.busi@u-paris.fr.
  • Grange W; Université de Strasbourg, CNRS, Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504, F-6700, Strasbourg, France; Université Paris Cité, UFR Sciences du vivant, F-75013, Paris, France. Electronic address: wilfried.grange@u-paris.fr.
Biochem Biophys Res Commun ; 731: 150370, 2024 10 30.
Article en En | MEDLINE | ID: mdl-39047619
ABSTRACT
Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN Idioma: En Revista: Biochem Biophys Res Commun Año: 2024 Tipo del documento: Article País de afiliación: Polonia Pais de publicación: Estados Unidos
Texto completo
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Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ADN Idioma: En Revista: Biochem Biophys Res Commun Año: 2024 Tipo del documento: Article País de afiliación: Polonia Pais de publicación: Estados Unidos