The Gateway cloning method is a method of molecular cloning invented and commercialized by Invitrogen since the late 1990s, which makes use of the integration and excision recombination reactions that take place when bacteriophage lambda infects bacteria. This technology provides a fast and highly efficient way to transport DNA sequences into multi-vector systems for functional analysis and protein expression using Gateway att sites and two proprietary enzyme mixes called BP Clonase and LR Clonase. In vivo, these recombination reactions are facilitated by the recombination of attachment sites from the lambda/phage chromosome (attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites (attLeft and attRight). The removal of the phage from the bacterial chromosome and the regeneration of attP and attB sites can both result from the attL and attR sites recombining under specific circumstances.
DNA sequences of interest are added to modified versions of these special Gateway Att sites. Two enzyme reactions take place, BP Clonase and LR Clonase. The BP Clonase occurs between the attB sites surrounding the insert and the attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme mixture and produces the entry clone containing the DNA of interest flanked by attL domains. As a byproduct of the reaction, the lethal ccdB gene is excised from the donor vector. The LR Clonase occurs between the attL regions of the generated entry clone and the attR regions of the target vector and is catalyzed by the LR Clonase enzyme mix. As a result, an expression clone with DNA of interest flanked by attB regions is produced. As in the BP reaction, a DNA sequence containing the ccdB gene is cut from the target vector.
Large archives of Gateway Entry clones, containing the vast majority of human, mouse, and rat ORFs (open reading frames) have been cloned from human cDNA libraries or chemically synthesized to support the research community using NIH (National Institutes of Health) funding (e.g. Mammalian Gene Collection, http://mgc.nci.nih.gov/ Archived 2015-02-25 at the Wayback Machine). The availability of these gene cassettes in a standard Gateway cloning plasmid helps researchers quickly transfer these cassettes into plasmids that facilitate the analysis of gene function. Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time and offers simpler and highly efficient cloning for downstream applications.
The technology has been widely adopted by the life science research community especially for applications that require the transfer of thousands of DNA fragments into one type of plasmid (e.g., one containing a CMV promoter for protein expression in mammalian cells), or for the transfer of one DNA fragment into many different types of plasmids (e.g., for bacterial, insect, and mammalian protein expression).