Denaturation of Streptavidin-Biotin bound dsDNA into ssDNA

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Mon Apr 12 06:26:23 EST 2010


As a possible improvement to Tom's approach, do a preceding PCR with a
much larger product by using an upstream forward primer.

That way, your biotinilated primers will bind halfway along the
template, become extended to the length of the preceding PCR, but any
remaining bound template will be easily separated on a gel when it
comes to purification whereas single-stranded DNA will appear as a
lighter band.

i.e: PCR1 on raw template:
   F1>
============================
                                           <R1

Product:
F1===================R1

PCR2 with preceding product as template:
         [Bt]-F2>
F1===================R1

SS Product size Vs. Template size Vs Product/Template Hybrids;
F1===================R1
F1-------[Bt]-F2=========R1 - Biotinylated product bound to template
           [Bt]-F2----------------R1

The mobility of single stranded DNA vs. semi-single-stranded DNA
versus double-stranded DNA, I don't know about. On the one hand, ssDNA
has the same charge proportionally to its mass, but its length (and
likelihood to be obstructed by gel fibres) remains the same. Hopefully
someone more experienced in ssDNA gels can jump in here.

Alternatively, you might be able to denature your DNA using alkaline
conditions. I suspect that's what the ammonium hydroxide approach is
intended for. Whether this will degrade the sepharose, break the
biotin bond or otherwise screw up the DNA is beyond my ken to predict
at present. I imagine it'd work, and from what I have seen there are
ways of tuning your alkalinity precisely to specific sequences so that
double-strands are broken based on their G-C content, etc.

Perhaps you can keep us posted on your progress here, I'd like to know
which method you use and which works!

On 12 April 2010 06:57, Tom Knight <tk from csail.mit.edu> wrote:
>
> 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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