A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation.
Golden Gate assembly
Intramolecular ligation
ssDNA synthesis
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
10 2023
10 2023
Historique:
revised:
26
05
2023
received:
02
12
2022
accepted:
05
06
2023
pmc-release:
01
10
2024
medline:
15
9
2023
pubmed:
27
6
2023
entrez:
27
6
2023
Statut:
ppublish
Résumé
Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.
Identifiants
pubmed: 37366288
doi: 10.1002/bit.28471
pmc: PMC10527171
mid: NIHMS1908527
doi:
Substances chimiques
DNA, Single-Stranded
0
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
3057-3066Subventions
Organisme : NIGMS NIH HHS
ID : R21 GM129559
Pays : United States
Informations de copyright
© 2023 Wiley Periodicals LLC.
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