Enzyme to digest dsDNA in ss/ds mix

Aawara Chowdhury aawara at nohome.org
Thu Jan 19 12:08:32 EST 2006

In <1137688772.541611.204820 at g44g2000cwa.googlegroups.com>,
 Susan Hogarth <hogarth at gmail.com> wrote:

>> If you used Taq (or any enzyme without proofreading ability), exoIII will
>> not digest the dsDNA PCR products, because Taq adds a 3' non-templated
>> nucleotide (usually dA) at the end.  exoIII only acts from blunt ends or
>> 5' overhangs.
> I used Klentaq; the Klentaq FAQ says:
> "Klentaq1 does put the extra A on. To do this efficiently, however,
> takes an extra long last extension (20-30 minutes)."

Then it sounds as if it is certainly worth trying an ExoIII digestion.  

However, I would suggest a digestion with lambda exonuclease over an ExoIII

Lambda exonuclease is a 5'->3' processive exonuclease.  It selectively digests
the strand that has a phosphorylated 5'-end.  This makes it particularly
useful to make single-stranded DNA (of a particular strand) after a PCR.  Its
activity on ssDNA and the non-phosphorylated strand of dsDNA is really poor.

Do a PCR with one phosphorylated primer, and one non-phosphorylated primer 
(Usually have the non-phosphorylated primer in about 3 - 4 fold excess
over the phosphorylated primer).  The PCR will result in two types of products:


and      ---------------------5'-OH

Digesting with lambda exonuclease will leave:




because it doesn't act efficiently on ssDNA, and on dsDNA starts digesting
from the phosphorylated end.

In America, through pressure of conformity, there is freedom of choice,
but nothing to choose from - Peter Ustinov.

More information about the Methods mailing list