PCR Mutagenesis-Based Method for Generation of Positive Controls for SSCP Analysis

We have developed a primer-mediated PCR mutagenesis-based method for the generation of positive controls to test the sensitivity of single-strand conformation polymorphism (SSCP) or any other PCR-based mutation screening method. This technique is based on the incorporation of a third longer primer, containing a mismatched base, into the PCR along with the two wild-type primers normally used to amplify DNA fragments for SSCP analysis. The longer mismatch primer (LMP) shares the sequence of one of the wild-type primers and also contains 5 to 10 additional bases, which include the mismatched base. The resulting PCR product is identical in length and sequence to the wild-type template with the exception that the LMP base mismatch is incorporated into nearly 100% of the product. We have observed an altered SSCP mobility pattern in all cases where positive controls have been generated using this technique. We believe that the use of such in vitro-generated controls can contribute to the interpretation of band patterns and to the optimization of experimental conditions for SSCP to facilitate maximum detection of sequence variants.