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
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