GATEWAY Cloning Technology is a universal system for cloning and sub cloning DNA fragments—universal in that all manner of DNA fragments can be cloned (cDNAs, genomic DNA, PCR fragments) into virtually any kind of vector (expression, sequencing, viral, plasmid). And once present in the GATEWAY system, a sequence can be transferred into any other GATEWAY vector while maintaining reading frame and orientation by simply adding proteins, incubating, and transforming E. coli. In addition, virtually any standard vector can be readily made GATEWAY compatible. The GATEWAY Cloning Technology is based on the site-specific recombination system used by phage l to integrate its DNA in the E. coli chromosome. Both organisms have specific recombination sites called attP in phage l site and attB in E. coli. The integration process (lysogeny) is catalyzed by 2 enzymes: the phage l encoded protein Int (Integrase) and the E. coliprotein IHF (Integration Host Factor). Upon integration, the recombination between attB (25 nt) and attP (243 nt) sites generate attL (100 nt) and attR (168 nt) sites that flank the integrated phage l DNA. Applications 1. Allows subcloning from one vector backbone to another. 2. Every subcloning reaction maintains the appropriate reading frame. 3. Subcloning process is fast and facilitates reaction automation. 4. Supports site specific recombination. 5. Multiple genes can be transferred to one or more vectors in one experiment. The assistance of Aqief Afzal, Research Assistant of ASRBC is highly appreciated.
Prof. Lutfur Rahman