[Q] PCR gives extra band - heteroduplex formation?
pnorton at lac.jci.tju.edu
Mon Oct 16 17:36:17 EST 1995
In article <1995Oct16.030355.21846 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) 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
> templates there is a third band of slower mobility (equivalent to ca. 400 bp)
> Does anyone know of a procedure (apart from cloning and sequencing)
> that would allow confirmation of this? - either a change to the PCR or the
> electrophoresis conditions would be ideal.
> Also, can anyone point me to an explanation as to how heteroduplex
> formation occurs and gives rise to this electrophoretic pattern? I have
> seen this explanation for extra bands given in the literature but no details
> were provided. I would also be interested in any alternative explanations
> for the pattern I see.
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. We have often seen such
artifacts in doing RT-PCR of transcripts with primers that span regions of
alternative splicing. Anecdotally, I have observed that a couple of freeze
thaw cycles can "resolve" the heteroduplexes - the extra bands just
disappear - so you might want to try this first.
As to the second question, I presume that the heteroduplexes only
appear later in the reaction, when the concentration of the both products
are high and they compete with primers for binding to each other. Thus,
you certainly might want to try lowering your cycle number. The structures
of the heteroduplexes are likely to be either Y-shaped or double strands
with bubbles. In either case, the single stranded regions cause altered
(generally reduced) mobility.
One final note: if your extra band is a heteroduplex, you can't
clone them, and sequencing only gives a mixture of the two authentic
products. I wasted a lot of time numerous years ago on trying to clone
and/or sequence a "band" that proved to be a heteroduplex.
Hope this helps,
Pamela A. Norton, Ph.D. Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107 p_norton at lac.jci.tju.edu
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