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Portrait of Jonas Tegenfeldt. Photo: Kennet Ruona

Jonas Tegenfeldt

Professor, Coordinator Nanobiology & Neuronanoscience

Portrait of Jonas Tegenfeldt. Photo: Kennet Ruona

Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51

Author

  • Karolin Frykholm
  • Camilla Freitag
  • Fredrik Persson
  • Jonas Tegenfeldt
  • Annette Graneli

Summary, in English

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity. (C) 2013 Elsevier Inc. All rights reserved.

Department/s

  • Solid State Physics
  • NanoLund

Publishing year

2013

Language

English

Pages

261-268

Publication/Series

Analytical Biochemistry

Volume

443

Issue

2

Document type

Journal article

Publisher

Elsevier

Topic

  • Condensed Matter Physics

Keywords

  • Single molecule
  • DNA
  • Rad51
  • Microfluidics
  • Supported lipid bilayer
  • Fluorescence microscopy

Status

Published

ISBN/ISSN/Other

  • ISSN: 1096-0309