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Tuesday, August 23, 2011

Gateway Cloning

Gateway® Cloning is a universal cloning technique developed by Invitrogen life technologies. Gateway® Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. It has effectively replaced the use of restriction endonucleases and ligases. Using Gateway®, one can clone/sub clone DNA segments for functional analysis.

Gateway® Cloning Mechanism

(a) The BP Reaction (PCR fragment + Donor vector = Entry Clone)

The BP Reaction is a recombination reaction which is explained in the following lines. For the reaction to take place, the gene of interest is amplified with the help of an attB tagged primer pair. The donor vector includes attP sites. The PCR product that includes the attB sites combines with the donor vector that includes the attP sites resulting in the formation of an entry clone. This integration reaction between the attB and the attP sites forms the basis of this reaction. The resulting entry clone contains the gene of interest flanked by attL sites.

(b)The LR reaction (Entry Clone + Destination Vector = Expression Clone)

The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. The entry clone generated from the BP reaction includes the attL sites. The Destination vector is designed to include the attR sites. The LR reaction is carried out to transfer the sequence of interest to one or more destination vectors in simultaneous reactions, making the technology high throughput. The entry clone is mixed with the appropriate Gateway® vector and Gateway® Clonase enzyme. Recombination between these sites generates two molecules. One molecule contains the DNA segment of interest, the other molecule is a by-product.

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.


Source:
http://www.premierbiosoft.com/tech_notes/Gateway_Cloning.html
http://en.wikipedia.org/wiki/Gateway_Technology