[Q] PCR gives extra band - heteroduplex formation?
hepad at ttu.edu
Tue Oct 17 08:17:36 EST 1995
Heteroduplexes are a frequent product of competitive RT-PCR reactions
which proceed into the plateau phase. In our experience, the
electrophoretic mobility of the heteroduplex has been very similar to
that of the larger of the two homoduplex products. So we have
tentatively concluded that the effective surface area of the
heteroduplex is determined by the larger single strand. We have
performed reactions with a number of different sized products and have
not been able to show a distinct third band.
How do we know we have heteroduplexes then?? We are using a reversed
phase HPLC technique in which retention is related to the number of
paired bases. Heteroduplexes always elute slightly faster than the
smaller of the two homoduplex products, indicating some loss of base
pairing at either side of the "bubble". In our situation we can
positively confirm that these products are heteroduplexes because we can
do reactions in which only one or the other product is produced. Then we
mix the reactions, melt and reanneal and the third product "appears".
Based on my experience, I would be suspicious that a reaction which
produces a possible heteroduplex which migrates so much slower on gel
electrophoresis is actually producing a non-specific product rather than
a heteroduplex. The difference in migration time seems difficult to
account for otherwise. However, HPLC should resolve this question. If
Bernard's apparent 400bp product is a heteroduplex it will elute faster
than it apparent "size". If it is a homoduplex it will elute true to its
size. The retention of double stranded DNA in the HPLC system is highly
In case you are interested, the HPLC column is from Sarasep (408 492
1029) and is called a DNASep column. If you are interested in this
technique and have access to gradient HPLC instrumentation I will be
happy to advise you on set up and solvent conditions. Email for details.
Peter A. Doris, Ph.D.
Associate Professor of Cell Biology and Biochemistry
Texas Tech University Health Sciences Center
More information about the Methods