PCR artifact induced by a base change in a primer

Gary Truett GaryTruett at POSTOFFICE.PBRC.EDU
Sat Sep 21 10:39:16 EST 1996

We have experienced a peculiar PCR artifact and are seeking comments from
others who may have had similar results.  Any references that address the
problem would be appreciated.

Our objective is to develop assays for genotyping mice that carry a
particular mutation.  The mutation affects no restriction sites, so we chose
to create a restriction site by altering a single base in one of the
primers.  We used GCG to identify a single base change that would generate a
restriction site that occurs in the wildtype DNA, but is lost in the mutant

PCR amplification generates a single product of the appropriate size.
The wildtype DNA should cut to completion, but only cuts partially.
Even more surprising is that the mutant DNA, which should not be cut at all,
cuts partially.

We have ruled out contamination of the reagents as a cause.

We have also ruled out the restriction endonuclease as the culprit.

Furthermore, we get similar results with different primers that amplify
completely different targets.

Lowering annealing temperature sometimes appears to resolve the problem.

Our current theory is that the base substitution in the primer causes the
wrong base to be incorporated at the site of the mutation (which is 1 to 4
bases away).

Is anyone familiar with this problem?

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