Enzyme to digest dsDNA in ss/ds mix

Susan Hogarth hogarth at gmail.com
Thu Jan 19 12:51:19 EST 2006


Aawara Chowdhury wrote:
> 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.

Ah, that's excellent, because then I would recover the ss product
*from* the ds product, as well as just the ss product itself. In fact,
that seems like it would remove the need for aPCR at all, really.

For aPCR, I have gotten better yield by increasing the ration - highest
ried (50:1) has been best so far. But that is not using phosporylated
primers.

- Susan



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