Easier Gene Cloning With Positive Selection Vectors |
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Nick Oswald |
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Isn’t it a pain digesting, purifying and dephosphorylating your cloning vector prep to eliminate prevent high background in your ligation/transformation? A new generation of positive selection vectors promises to eliminate all of that hassle by killing off any vector that has not taken up the insert you are trying to clone.
Positive selection vectors are currently available from Pure Biotech, Fermentas and Genhunter to name a few. The positive selection comes from a conditionally expressed lethal gene, such as a restriction enzyme, whose coding sequence contains the multiple cloning site. The lethal gene is disrupted by the ligation of a DNA insert into the multiple cloning site. Any undigested or self-ligated vector copies retain a functional copy of the lethal gene and are therefore selected against in the transformation.
This eliminates the need for extensive vector purification, and in fact the manufacturers say that no vector preparation is required at all – just digest, ligate and transform without any purification at all and get close to 100% positive clones back. Sounds great – I think I might try it out!
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Ligation Independent Cloning Protocol
Ligation Independent Cloning Primer Design
Sequence and Ligation Independent Cloning
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Positive selection vectors are helpful in many cases, but there are two potential disadvantages to be aware of. First, in some cases it is possible to transcribe through the insert or to re-initiate transcription within the insert. As a result, the toxic protein will still be expressed, and the clones will be lost. These problems are more likely with small inserts or when cloning promoters. The second problem is that the insert will be transcribed by the promoter that drives expression of the lethal gene. This expression can lead to difficulty in cloning ORFs, toxic peptides, or regions of high secondary structure.