Ligation of alkaline phosphatase treated DNA

Andy Bates bates at liv.ac.le
Mon Mar 6 10:17:55 EST 1995


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

> A.p. is used to prevent ligation of multiple inserts in one vector by 
> dephosphorylation of the 5'terminis of the 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).

Dietmar,

I'm pretty sure your 'strange theory' is correct.  When the plasmid
ends up in the cell, DNA polymerase I, for example will repair the
missing phosphate by nick translation, followed by ligation.

Andy.
____________________________________________________________
Dr. Andrew D. Bates             Tel:   (+44) (0)151 794 4322
Department of Biochemistry      Fax:   (+44) (0)151 794 4349
University of Liverpool         Email: bates at liv.ac.uk
PO Box 147
Liverpool
L69 3BX, UK



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