The Gateway cloning System, invented and commercialized by Invitrogen since the late 1990s, is a molecular biologymethod that enables researchers to efficiently transfer DNA-fragments between plasmids using a proprietary set of recombination sequences, the "Gateway att" sites, and two proprietary enzyme mixes, called "LR Clonase", and "BP Clonase". Gateway Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. Using Gateway, one can clone subclone DNA segments for functional analysis. The system requires the initial insertion of a DNA fragment into a plasmid with two flanking recombination sequences called “att L 1” and “att L 2”, to develop a “Gateway Entry clone”. Large archives of Gateway Entry clones, containing the vast majority of human, mouse and rat ORFs have been cloned from human cDNA libraries or chemically synthesized to support the research community using NIH funding. 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 down-stream 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, or for the transfer of one DNA fragment into many different types of plasmids.
Basic Steps
The first step in Gateway cloning is the preparation of a Gateway Entry clone. Entry clones are often made in two steps: 1) “Gateway attB1, and attB2” sequences are added to the 5’, and 3’ end of a gene fragment, respectively, using gene specific PCRprimers and PCR-amplification; 2) the PCR amplificationproducts are then mixed with special plasmids called Gateway “Donor vectors” and the proprietary “BP Clonase” enzyme mix. The enzyme mix catalyzes the recombination and insertion of the att-B-sequence-containing PCR product into the att P recombination sites in the Gateway Donor vector. Once the cassette is part of the target plasmid, it is called an "Entry clone" in the Gateway nomenclature, and recombination sequences are referred to as the Gateway “att L” type. The gene cassette in the Gateway Entry clone can then be simply and efficiently transferred into any Gateway Destination vector using the proprietary enzyme mix, “LR Clonase”. Thousands of Gateway Destination plasmids have been made and are freely shared amongst researchers across the world. Gateway Destination vectors are similar to classical expression vectors containing multiple cloning sites, before the insertion of a gene of interest, using restriction enzyme digestion and ligation. Gateway Destination vectors are commercially available from Invitrogen, EMD and Covalys. Since Gateway cloning uses patented recombination sequences, and proprietary enzyme mixes available only from Invitrogen, the technology does not allow researchers to switch vendors and contributes to the lock-in effect of all such patented procedures. To summarize the different steps involved in Gateway cloning:
Gateway BP reaction: PCR-product with flanking att B sites + Donor vector containing att P sites + BP clonase => Gateway Entry clone, containing att L sites, flanking gene of interest
Gateway LR reaction: Entry clone containing att L sites + Destination vector containing att R sites, and promoters and tags + LR clonase => Expression clone containing att B sites, flanking gene of interest, ready for gene expression.
