Golden EGG, a simplified Golden Gate cloning system to assemble multiple fragments

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 a...

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Bibliographic Details
Published in:Scientific reports 2024-10, Vol.14 (1), p.25288-12, Article 25288
Main Authors: Biró, János Barnabás, Kecskés, Kristóf, Szegletes, Zita, Güngör, Berivan, Wang, Ting, Kaló, Péter, Kereszt, Attila
Format: Article
Language:English
Subjects:
631/1647
631/326
631/337
631/449
Cloning
Cloning vectors
Cloning, Molecular - methods
Deoxyribonucleic acid
DNA
DNA - genetics
DNA - metabolism
DNA Restriction Enzymes - genetics
DNA Restriction Enzymes - metabolism
Enzymes
Genetic Vectors - genetics
Humanities and Social Sciences
multidisciplinary
Polymerase Chain Reaction
Science
Science (multidisciplinary)
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-024-77327-4