PCR primer mismatches

Dale R Shepard dshepard at magnus.acs.ohio-state.edu
Tue Nov 29 12:11:53 EST 1994


I am having a problem with an upstream primer which binds to a secondary site 
on my template DNA.  In reactions with both upstream and downstream primers and
template DNA, I get a band smaller than my expected 1.8 kb (this is the only 
band).  I get the same band with only the upstream primer and the template DNA.
I have tried three different primers and always have this occur (with different
sizes pieces for each primer).  I am using Perkin Elmer AmpliTaq polymerase and
I have used a couple of different reaction conditions.  My reactions have 
contained 0.25 uM or 1 uM primers, 2.5 mM Mg chloride and 0.2 mM dNTPs.  My 
temperature programs have been with a 45 C annealing temperature or a 55 C 
annealing temperature during each amplification cycle, or with a combined 72 C 
annealing/extension for each cycle.  All these conditions have given me the 
same result.  My primers have been 19-, 24- and 27-mers.  Comparing the primer 
sequences to the template DNA with the Fasta command on GCG, the binding site I
want is about 87-95% similar to the template over the whole length of the 
primer, while the secondary sites are about 90 % similar to a 7-10 bp overlap.
Should I change my primers again? (I'm trying to introduce a BamHI site), use 
an enhancer (like the one Stratagene sells), or change my reaction conditions?
Any help would be appreciated.  Sorry for the length of the message, but I 
didn't want to cause confusion.  Thanks in advance, I can post a summary of the
responses.



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