[Q] PCR gives extra band - heteroduplex formation?
bernard at elsie.nci.nih.gov
Sun Oct 15 22:03:55 EST 1995
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)
than that of the largest. This is true of 12 individual samples of each
type (one, other, both). I believe that this phenomenon can be seen as a result
of heteroduplex formation.
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.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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