PCR artifact induced by a base change in a primer

brett brett at BORCIM.WUSTL.EDU
Sat Sep 21 16:17:01 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
>DNA.  
>
>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?

Sure, I have seen cloned PCR products with errors in the primed region. The
chemistry of primer synthesis can lead to all kinds of errors, but misincorp-
oration is relatively rare. Internal deletions and incomplete synthesis is
more common. You could always gel purify your primers. Double check the
order and spec sheets for proper sequence input, if they are wrong
re-synthesize it. Otherwise, pick more clones to begin with and sequence
through the primed DNA.


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             

"I own my own pet virus. I get to pet and name her." - Cobain




More information about the Methods mailing list