Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Does a restriction enzyme cut at a related site? - (Jul/29/2014 )

I have a silly confusion.I wanted to know whether a restriction enzyme can recognize a incompatible site with compatible sticky endand cut it?


For e.g, SpeI and XbaI can generate compatible sticky ends but a slightly different recognition sites


For XbaI recognition site is TCTAGA and it cuts between T and C to generate T            CTAGA

                                           AGATCT                                                                 AGATC            T


For SpeI recognition site is ACTAGT and it cuts between A and C to generate A            CTAGT

                                           TGATCA                                                                 TGATC             A

So can SpeI cut XbaI sites and vice versa under any conditions?
So like the above example is this possible at all.If it happens I can have serious problems while cloning.
When SpeI and XbaI generate compatible sticky ends and they ligate,will the resulting mixed site be recognized and cut by either of the two enzymes?
Please can anyone clear this for me.unsure.png


If you cut first one with XbaI and second with SpeI


Then the first one is originaly:




And second is originaly:





Then after cut and ligation you will have:





which is neither XbaI or SpeI recognition sequence, and not even a palindrome. So none of these will be able to recut.


Depending on the surrounding sequence, you may be able to find enzyme that cuts this with the same overhang, using a NEB Enzyme Finder, one of the enzymes that have ambiguitious recognition sites. But they will not cut as specifically as XbaI or SpeI.


As Trof stated, the mixed site should not be recognized.  The use of compatible overhangs to destroy sites at ligation is the foundation of some modular construct approaches such as BglBrick (which uses ligation of BglII and BamHI).  I've used this extensively and have never had cutting of the mixed sites.  What sort of cloning problem are you having?  Could you be a bit more specific?


I suppose that he actually needs the mixed site to be recut by XbaI or SpeI.


Thank you for your replies.But nobody cleared the confusion as to whether SpeI and XbaI can recognize and cut each other's sites? Is it possible due to Star Activity?


Neither XbaI nor SpeI have a reported star activity in any of the NEB buffers, so it is unlikely.

In any case, star activity, if exhibited is unpredictible and low-efficient, though some conditions can increase it's possibility. But as I understood it, only in enzymes that are prone to it. 


If you need both vector and insert to be cut by same enzyme, you may use the PCR approach and PCR the smaller (insert) with a proofreeding polymerase and primers with overhang designed to contain the modified restriction site and few additional bp, then cut the PCR product and ligate. This approach was heavily discussed through the forums.