Overview of Gateway Technology
The cloning and manipulation of DNA has historically relied upon the use of “cutting and pasting” techniques to bring together the required pieces of DNA into a clonable form. For example, to perform a simple cloning of a PCR product into an existing vector might involve the following steps:
- Purifying a large amount of the vector of choice (getting enough raw material to work with)
- “Cutting” the vector with one or more restriction enzymes (producing one part of the final clone)
- Dephosphorylating the required piece and purifying it away from any unwanted pieces
- Obtaining the second piece of the final vector (via PCR, and/or some combination of restriction digestion…)
- Using a ligase to connect the two pieces of DNA (the pasting step)
- Transforming the ligated fragments into E. coli
- Selecting the clones of interest using antibiotic resistance and/or some-other properties…
The problem with these steps is that they are very time consuming. Often the screening and recovery of suitable clones is problematic: many of the clones recovered may be unwanted by-products or co-products of the process. Whilst molecular biological reagents are very expensive, often it is the amount of time and labour required that represents the major expense in such a methodology.
Gateway technology provides a “short cut” to the above procedure. Particularly it removes most of the requirements for the use of restriction and ligation enzymes and vastly simplifies the recovery of positive clones. The equivalent steps in a Gateway cloning of a PCR product would be:
- Obtain a small amount of a pDONR vector containing attP sites (large amounts not required, cutting not required)
- PCR the target of choice using PCR primers with specific recombination sequences present (attB sites).
- Mix the vector, PCR product and the recombinase mix and allow to react.
- Transform into an appropriate E. coli strain.
- Select the clones of interest using antibiotic selection (typically >90% of clones recovered are correct)
How this works can be demonstrated by looking at the following example of a Gateway vector and understanding the function of the various genetic components: