Gateway Technology - Basic Steps

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 PCR primers and PCR-amplification; 2) the PCR amplification products are then mixed with special plasmids called Gateway “Donor vectors” (Invitrogen nomenclature) 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 (Invitrogen nomenclature for any Gateway plasmid that contains Gateway “att R” recombination sequences and elements such as promoters and epitope tags, but not ORFs) 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 (Novagen) 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 (e.g., amplified from cDNA library) + Donor vector containing att P sites + BP clonase => Gateway Entry clone, containing att L sites, flanking gene of interest
  • this step can be replaced by other cloning methods
• 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.