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Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthetic biology company, Telesis Bio.
The Gibson assembly method is a relatively straightforward DNA assembly method, requiring only a few additional reagents: the 5' T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The DNA fragments to be assembled are synthesised to have overlapping 5' and 3' ends in the order that they are to be assembled in.
This is the main advantage of OE-PCR and other long-homology based cloning methods such as Gibson assembly, which overcome the limitations of traditional restriction enzyme digestion and ligation cloning methods. [3] Assembly of custom DNA sequences with OE-PCR consists on three main steps.
Gibson assembly is a quick cloning method that uses three primary enzymes; 5' exonuclease, polymerase and ligase. [15] The exonuclease digests the 5' end of DNA fragments leaving a 3' overhang. [15] If there is significant homology (20-40 bp) on each end of the DNA insert, it can anneal with a complementary backbone. [15]
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Golden Gate assembly involves digesting DNA sequences containing a type IIS restriction enzyme cut site and ligating them together. Golden Gate Cloning or Golden Gate assembly [1] is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. [2]