How to generate sticky ends? - (Mar/04/2010 )

I am a rookie in PCR. How to generate sticky ends at both sides of my PCR products without the using of restriction enzymes? Thanks.

If you use primers with a U instead of a T in a specific location, then pcr (using taq, not proofreading enzymes) then you can remove the u and 5' portion of the primer enzymatically. See the NEB USER enzyme kit.

You can also make primers with a 2' methoxy modified base, which will halt pcr enzymes leaving a 5' overhang. You need sufficient bases 3' of the modified base to bind to the template.

This may not be the approach you are looking for, but it's worked great for me when I wanted to add sticky ends to my PCR products: run your PCR as you always would and clone it into a TOPO cloning vector. You can then restriction cut your PCR product from the TOPO cloning vector using any of the restriction enzymes present in the vector. The big advantage of this approach is that all you need is a single TOPO clone to work and you will not have to PCR amplify your fragment ever again since you can grow the TOPO clone (containing your fragment) in bacteria.

You can also make primers with a 2' methoxy modified base, which will halt pcr enzymes leaving a 5' overhang. You need sufficient bases 3' of the modified base to bind to the template.