Exonuclease VII vs Mung Bean Nuclease

Dom Spinella dspinella at chugaibio.com
Thu Aug 6 09:43:51 EST 1998

> Having heard about these undesireful effects of mung bean nuclease I had
> to try it recently myself, chewing back two 4bp non-sticky 3'-overhangs,
> and blunt-end ligate those back in order to get the right reading frame.
> Surprise, surprise: none of the clones sequenced had any DNA chewed
> back and 12 out of 16 were of the desired sequence. The others were
> uncomplete digestions of mung bean, creating a 2bp sticky overhang that
> got ligated (and therefore getting the thing out of RF).
> So, in the end, I think MBN works nicely. Just control it well: I made the
> MBN incubation at RT and made serial dilutions of the enzyme followed by
> phenol/chloroform extraction and precipitation. Ligated each of the
> dilutions separately and choosed clones from the highest MBN
> dilution giving significantly higher numbers of colonies (about 10 x)
> compared to the non-MBN control (or the vector only control if using MBN
> during cloning).
> Good luck, jorg

Please don't misunderstand my earlier postings -- I did not mean to
imply that MBN will not work at all for polishing single-stranded
overhangs.  It surely does.  However as you note, one needs to control
the reaction carefully (for example, by making serial dilutions of MBN
as you have done).  At best, this entails a bit more work than using
Klenow or T4 Pol to do the same job. Moreover, if 25% of your clones
were derived from incomplete MBN digestions in which the overhangs were
repaired by the bugs, you can bet that there were many more such
reaction products that failed to clone altogether. This also suggests
that your "optimal" MBN concentration was pretty low, so its not
surprising that you didn't see any "chewback" reaction products. 

Frankly, if all you need is one clone, almost anything will work.  The
relevant comaprison is the number of correct clones obtained per unit
input of DNA when polished by MBN vs Klenow or T4 Pol.  I suspect that
the polymerases are more efficient (you might have seen 20 or 30X over
background for example) -- particularly when the overhang is at the 5'
end as with the original posting. This "gut feeling" is derived not only
from conventional wisdom as dispensed in the popular cloning manuals and
literature, but from experience with producing cDNA libraries by adding
adapters to MBN or T4 polished cDNA. I have always obtained many more
clones after switching to T4 Pol (from MBN) to polish the cDNA although
I have never compared the polishing techniques directly with the same
constructs. Has anyone out there ever made such a head-to-head
comparison? Do let us know the results. Cheers. --Dom Spinella

More information about the Methods mailing list