[Q] PCR gives extra band - heteroduplex formation?

[Robert Horton]

Bernard Murray (bernard at elsie.nci.nih.gov) wrote:
:         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. 

If I understand correctly, you are co-amplifying two related molecules;
one has an insert, and one does not:

         >---------------->
         <----------------<
                 and
     >--------++++++++-------->
     <--------++++++++--------<

Heteroduplexes would be molecules like this:

         >---------------->                  ++
         <-------||-------<                 +  +
                 ++           and           +  +
                +  +                         ++
                +  +                 >-------||------->
                 ++                  <----------------<

where one insert-free strand and one insert-containing strand hybridize 
to one another, and the insert loops out. Such a molecule would be 
expected to have weird migration characteristics, since it is part 
single-stranded loop.

These heteroduplex molecules would be expected to form if the 
concentrations of each strand were high enough for them to compete 
kinetically with the primers for annealing. In other words, at low 
product concentrations, the primers would be expected to bind to the DNA 
strands much more quickly than product strands could find one another. 
Fill-in results in "pure" molecules, not heteroduplexes. At high product 
concentrations, strands that have formed in previous cycles can 
hybridize to one another fast enough to compete with the primers, and 
some of the resultant products will be heteroduplexes.

So basically, if you don't do as many cycles (i.e., stay at relatively 
low product-strand concentrations), you won't see as many 
heteroduplexes.



Robert M. Horton (PhD!) /\ "Crash programs fail because of the theory that
U of M Dermatology Dept || with nine women pregnant you get a baby a month"
Box 98 UMHC, 4-154 PWB /||\ -W. von Braun.   Disclaimer: "Bob who?"
Minneapolis, MN 55455   ^^   horton at biosci.cbs.umn.edu       (612)625-8941