PCR problem - please help

[Richard M Rohan]

Shahram Mori (smori at nmsu.edu) wrote:
: Vince Mulholland (mulholl at sasa.gov.uk) wrote:
: : I've got a contaminating band in my PCR reactions. I'm using 
: : allele-specific amplification to differentiate between species of 
: : nematodes. The set-up is as follows;

: :        'A'-specific         'B'-specific                    Universal
: :             -->                  -->                          <--
: :           390 bp               240 bp

: : So, the PCR reaction contains three primers. I get a particular band 
: : for each species or both bands when there is a mixture. This has been 
: : working fine up until a few weeks ago. I still get a single band when 
: : I amplify a single species. However, when there is a mixture of the 
: : two species I get a contaminating 800 bp band. This is the odd part - 
: : it is ONLY present when there is a mixture.

: : Anyone had similar problems? Any ideas gratefully accepted.

Souds like heteroduplex formation to me.  If the allele-specific products are
sufficiently similar they could be hybridizing in later cycles to form an
A:B heteroduplex.  Since this heteroduplex is partially single-stranded it
will migrate slower in the gel, thus giving the appearance of a larger
fragment.  Acrylamide gels are more susceptible to this problem than agarose
gels.

If you can detect the two allele-specific bands, why worry about the larger
fragment?

Rich Rohan
rrohan at world.std.com