Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
25 Oct 2024
25 Oct 2024
Historique:
received:
03
05
2024
accepted:
21
10
2024
medline:
26
10
2024
pubmed:
26
10
2024
entrez:
25
10
2024
Statut:
epublish
Résumé
The Golden Gate method is an efficient tool for seamless assembly of multiple DNA fragments, which uses Type IIS restriction endonucleases, cleaving the DNA outside of their recognition site to release DNA parts from PCR fragments or entry clones, thus allowing the design of overhangs for ligation at will. However, the construction of the entry clones requires the use of other restriction enzyme(s) or cloning techniques and different entry vectors for the individual overhangs. Here, we present a simplified Golden Gate cloning approach termed Golden EGG. It features (1) a single entry vector with a specific cloning site to host the DNA parts; (2) a unique primer design to create the restriction enzyme recognition site to release the fragments with the overhangs at will; (3) the use of a single Type IIS enzyme for the construction of both the entry and destination clones; (4) a specific temperature profile during the digestion-ligation reaction. Our user-friendly, streamlined method retains the key attributes of the Golden Gate technique, while offering the potential to generate compatible parts with any existing Golden Gate toolkit and to be accessible to a wide user base without the need for extensive acquisition of new vectors or expensive enzymes.
Identifiants
pubmed: 39455683
doi: 10.1038/s41598-024-77327-4
pii: 10.1038/s41598-024-77327-4
doi:
Substances chimiques
DNA
9007-49-2
DNA Restriction Enzymes
EC 3.1.21.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
25288Subventions
Organisme : Hungarian National Research, Development and Innovation Office
ID : K134841
Informations de copyright
© 2024. The Author(s).
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