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.


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

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

More information about the Methods mailing list