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Gateway Cloning is a universal cloning technique that facilitates the efficient transfer of DNA fragments between different cloning vectors without disrupting the reading frame. Developed by Invitrogen Life Technologies, this cloning system uses as a proprietary set of recombination sequences called the Gateway att sites, and two proprietary enzyme mixes - BP Clonase and LR Clonase.
The Gateway Cloning works through two distinct reactions:
The BP Reaction is a recombination reaction that takes place between the att B sites flanking the insert and the att P sites of the donor vector. This reaction generates the entry clone, which contains the DNA of interest flanked by attL sites. During the reaction, the ccdB gene is eliminated from the donor vector. The BP Reaction is catalyzed by the BP Clonase enzyme mix.
The LR Reaction is also a recombination reaction. This reaction takes place between the attL sites of the generated entry clone and the attR sites of the destination vector. It generates the expression clone with the DNA of interest flanked by attB sites. During the reaction, the ccdB gene is eliminated from the destination vector. The LR Reaction is catalyzed by the LR Clonase enzyme mix.
By circumventing limitations of traditional restriction enzyme cloning, Gateway cloning makes it possible to access virtually any expression system in just a few easy steps.