Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51
Frykholm, Karolin ; Freitag, Camilla ; Persson, Fredrik ; Tegenfeldt, Jonas LU
and Graneli, Annette
(2013)
In Analytical Biochemistry
443(2).
p.261-268
- Abstract
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4196484
- author
- Frykholm, Karolin
; Freitag, Camilla
; Persson, Fredrik
; Tegenfeldt, Jonas
LU
and Graneli, Annette
- organization
- publishing date
- 2013
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Single molecule, DNA, Rad51, Microfluidics, Supported lipid bilayer, Fluorescence microscopy
- in
- Analytical Biochemistry
- volume
- 443
- issue
- 2
- pages
- 261 - 268
- publisher
- Elsevier
- external identifiers
-
- wos:000327279700023
- scopus:84886741534
- ISSN
- 1096-0309
- DOI
- 10.1016/j.ab.2013.08.023
- language
- English
- LU publication?
- yes
- id
- af450762-6bce-436d-b0a9-7d36e64a8c4d (old id 4196484)
- date added to LUP
- 2016-04-01 10:08:28
- date last changed
- 2023-09-13 18:50:15
@article{af450762-6bce-436d-b0a9-7d36e64a8c4d,
abstract = {{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.}},
author = {{Frykholm, Karolin and Freitag, Camilla and Persson, Fredrik and Tegenfeldt, Jonas and Graneli, Annette}},
issn = {{1096-0309}},
keywords = {{Single molecule; DNA; Rad51; Microfluidics; Supported lipid bilayer; Fluorescence microscopy}},
language = {{eng}},
number = {{2}},
pages = {{261--268}},
publisher = {{Elsevier}},
series = {{Analytical Biochemistry}},
title = {{Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51}},
url = {{http://dx.doi.org/10.1016/j.ab.2013.08.023}},
doi = {{10.1016/j.ab.2013.08.023}},
volume = {{443}},
year = {{2013}},
}