Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
22 07 2022
22 07 2022
Historique:
accepted:
13
04
2022
revised:
18
03
2022
received:
14
08
2021
pubmed:
1
5
2022
medline:
26
7
2022
entrez:
30
4
2022
Statut:
ppublish
Résumé
Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.
Identifiants
pubmed: 35489063
pii: 6576366
doi: 10.1093/nar/gkac300
pmc: PMC9303394
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e77Subventions
Organisme : Arthritis Research UK
ID : FC001119
Pays : United Kingdom
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Wellcome Trust
ID : FC001119
Pays : United Kingdom
Organisme : Cancer Research UK
ID : FC001119
Pays : United Kingdom
Organisme : Medical Research Council
ID : FC001119
Pays : United Kingdom
Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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