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
digestion.

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:

      5'P---------------------
         ---------------------5'-OH

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

Digesting with lambda exonuclease will leave:

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

                    +

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

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

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


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