Allele-specific PCR amplification due to sequence identity between a PCR primer and an amplicon: is direct sequencing so reliable?

PCR-direct sequencing (DS) is thought to be a very reliable method of determining DNA sequence and genotyping. Under certain conditions, however, DS can generate inaccurate results. Here we report a case of erroneous DS, in which a single nucleotide polymorphism (SNP) in the human PAX9 gene was mistyped due to allele-dependent PCR amplification. Examination of the amplified region showed that the 5' eight bases of one of the PCR primers were identical to the eight bases of the reverse strand downstream of the SNP, and the ninth base matched one of the alleles. Altering the primer so that it matched the other allele reversed the allele-specific inhibition. Reducing the base-pairing abolished the inhibition. Thus, the SNP was responsible for the difference in annealing efficacy of the primer and was therefore critical for the allele dependency. The allele-specific inhibition presented here can occur with any PCR primer sequence that encompasses a site that is polymorphic in the gene sequence. This phenomenon needs to be considered as a possibility when interpreting results from all PCR-based experiments. Sequence similarity between PCR primers and internal amplified regions should be considered for all methods for mutation detection and genotyping using PCR.