Denaturation of Streptavidin-Biotin bound dsDNA into ssDNA

Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu)
Mon Apr 12 00:57:31 EST 2010


Guo Wei Kua <gwkboy from gmail.com> writes:
> 
> I have PCR products of about 70bp. One strand is biotinylated,
> because I used biotinylated forward primers. Now, how do I isolate
> only this strand?  Do I denature first, then use beads to capture
> the ssDNA? If so, how to do that? I am afraid that once I denature
> the dsDNA with heat, I won't be fast enough to capture the ssDNA
> before reannealing.  Or do I capture the dsDNA first, then
> heat-denature the dsDNA so that the non-biotin strand will fall off?
> The problems hereby is, again, reannealing of the DNA. Second,
> heat-denature may even break the biotin-bead bond, causing
> everything to be lost when I discard the sup? Help!!
> 
> PS: I prefer not to use high salt solutions, as the final purified
> ssDNA should not hv salt if possible.

I think this is the difficult approach.  Why not create just the
strand you want with linear amplfication using a single primer.  Add
just the biotinylated primer to a "PCR" reaction with large amounts of
template DNA (perhaps a previous pcr with both primers, diluted
significantly, say 100x).  Do cycling as normal, but expect linear
amplification with production of only the primed strand. Done, unless
you really need to eliminate ALL of the reverse strand.  Otherwise,
purify with biotin.  With this method you could probably work without
biotinylated primers at much lower cost.



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