[Q] PCR gives extra band - heteroduplex formation?

M. Alexeyev malexeyev at biost1.thi.tmc.edu
Mon Oct 16 20:18:11 EST 1995


In article <pnorton-1610951736170001 at norton1.jmc.tju.edu>,
pnorton at lac.jci.tju.edu (Pamela Norton) wrote:


> > Hello all,
> >         I am using PCR to co-amplify two genomic DNA fragments.  The
smallest
> > is ca. 200 bp and the larger contains an insertion in the middle of the
> > smaller of ca. 80 bp.  When I analyse the products by electophoresis (2%
> > agarose, x1 TBE) I see the appropriate sized band in samples in which
only one
> > (either) of the two templates was present.  However, in samples
> containing both
> > templates there is a third band of slower mobility (equivalent to ca.
400 bp)
> 
> Bernard,
> 
>      To answer the first question, if you radiolabel the PCR products in
> some fashion and run on a denaturing (urea) gel, you should have only the
> two bands corresponding to the predicted products..

>             Pam Norton
> 
> -- 
>


Guys,
I have an urge to ask you a silly question: what is an expected
electrophoretic  mobility of heterodimer? Base pairs-wise it should be at
appr. 240 bp (200 bases one strand + 280 bases another). Does that 80
base-long loop retard heterodimer to a mobility of 400 bp fragment? If so,
is there any way to predict the apparent mobility of heterodimers (say, in
this particular case we have 80-base loop that added 160 bp to an apparent
mobility of heterodimer. Therefore, we have 1:2 ratio). Sorry again.

Misha.



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