Sub-cloning sticky-end PCR products - (Jun/17/2009 )

Hi,

I am trying to modify a pcDNA3.1D/V5-His-TOPO vector (Invitrogen, K4900-01) to replace one insert with another. Originially, another insert (containing an N-terminal 3XFLAG tag) was sub-cloned into this vector by a colleague, following the kit protocol (i.e. using a 5' sticky-end/3' blunt-end PCR product for directional cloning). I want to cut out this insert but leave behind the 3XFLAG sequence for tagging my new insert.

I have found a restriction enzyme (RE) that will cut in the linker sequence between the 3XFLAG tag and the insert I want to remove, but this RE will also cut at the 5' end, thereby creating sticky-ends at both the 3' and 5' ends of the plasmid sequence. This is good because the insert I want to cut out will be removed, but I am not sure what effect having a 5' sticky-end in the plasmid will have on the subsequent cloning.

I want to generate a PCR product with a 5' overhang (complementary to the 3' sticky-end of the plasmid) for directional cloning, but my question is, can the 5' sticky-end of the plasmid (i.e. the cut where the 3' end of my insert needs to ligate to re-circularise the plasmid) be repaired, or will having the sticky-end product from the RE digest prevent re-formation of a circular DNA?

Btw, apologies if I am confusing the 5'/3' terms (I often do!).

Thanks for any help,
N.

There is a reason for your confusion on 3' vs. 5' ends. Each linear DNA fragment has a 3' and 5' end AT BOTH ENDS. Using those words to distinguish the ends of a DNA fragment makes no sense. You need a different way of identifying and discussing the ends.

If you cut your existing plasmid with a single RE, then it will religate and form a circular plasmid with no insert. If you add a linear fragment with compatible ends, it may insert into the vector, but will do so in a random orientation, since both ends are the same.

Can you cut your vector with the RE that you found as well as a second one which will leave a different end?

Thanks for the reply, and apologies for any confusion caused by my mis-use of 5' and 3' terms. You have made a very good point about the potential for the plasmid to re-circularise if I only cut with one restriction enzyme. I will look for a second restriction site and think about adding an overhang to my insert that is complementary to the second cut. Hopefully having two sticky-ends with different sequences will improve the chances of ligating my insert in the right orientation. Thanks again!

N.