3' overhang digestion

David F. Spencer dspencer at is.dal.ca
Thu Oct 23 13:36:57 EST 1997


In article
<Pine.SOL.3.91.971014114420.14448A-100000 at godzilla3.acpub.duke.edu>, Javier
Elbio Irazoqui <jei at acpub.duke.edu> wrote:

> I'm in need of a protocol for digestion of 3' overhangs. I don't have 
> mung bean nuclease, but I do have S1 nuclease. The overhang is 2 bp long. 
> I need to do this to clone this digestion fragment.
> Thanx

The usual method for this is treatment with T4 DNA polymerase, in the
presence of dNTPs. T4 DNA polymerase has a much more aggressive 3'->5'
exonuclease than the E. coli equivalent (the Klenow fragment of PolI) and
provided that is has adequate dNTPs will apparently chew back DNA to a
blunt end and then essentially  reach an equilibrium of removing the first
3' base from the ds DNA and replacing it immediately.  I seem to recall
that the polymerase:exo activity ratio is about 10:1 for T4 DNA polymerase
but about 100:1 for Klenow, which is why T4 DNA polymerase has such a high
replication fidelity.  A T4 DNA polymerase mix is a good general approach
for blunting any restriction digest, either 5' overhang or 3' overhang,
including presumably removing the 3' 'A' from PCR products generated with
3'-exonuclease minus polymerases such as Taq.  Throw some polynucleotide
kinase with some rATP into the soup and you will also phosphorylate the
normally 5'-OH terminated primers, thus allowing cloning into blunt
cut/phosphatased vector.

I wouldn't think either mung bean or S1 is a particularly good way of doing
what you want.

Dave

--------------------------------------------
David F. Spencer, PhD
Dept. Of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca



More information about the Methods mailing list