[Q] PCR gives extra band - heteroduplex formation?

[Peter Doris]

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 
size dependent.

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