Ligation of alkaline phosphatase treated DNA

David Micklem drm21 at mole.bio.cam.ac.uk
Mon Mar 6 11:56:24 EST 1995


In article <3ir2ka$mep at nz12.rz.uni-karlsruhe.de>,
uc05 at rzstud1.rz.uni-karlsruhe.de (Dietmar Baas) wrote:

> Dear Netters,
> 
> I have a theoretical problem with the ligation of DNA, treated with
> alkaline phosphatase (a.p.) and here is my question:
> A.p. is used to prevent ligation of multiple inserts in one vector by 
> dephosphorylation of the 5'terminis of the insert.

Oh!  This is exactly the opposite of what I use a.p. for.  (When I use
a.p.) I usually a.p.
the VECTOR so that it can't recircularize without incorporating an insert.
The reasoning behind this is that intramolecular  (ie single molecule) ligation
should be favoured over intermolecular ligation (ie vector to insert). 

 But all books say,
> T4 DNA Ligase needs 5'phosphate and 3'OH. This results in a gap in one
> of the two strands. How is this gap closed? ATP is present in the 
> ligation mixture but I never heared something about kinase activity 
> of T4 DNA Ligase.
> Another possibility is the "in vivo repair" of the gap after transformation
> by enzymes of E.coli (but this sounds like a strange theory).

I think that strange as it sounds, this is exactly what happens (but I
could be wrong)!

Cheers,

David

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D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
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