Aim To analyze the specificity of the on/off switch mediated by high fidelity proofreading DNA polymerases and 3'-phosphorothioate-modified allele-specific primers for screening certain sites of single nucleotide polymorphism (SNP) in genomic DNA. Methods The genomic DNA is used as the template. The 3'-phosphorothioate-modified allele-specific primers were evaluated in their effects on primer-extension with genomic DNA harboring SNP sites allele. Results The products from Taq had many mixed bands, and some of them clearly showed false positives. However,at most of the situations, high fidelity DNA polymerase of Pfu yielded single products. Conclusion These data suggest that the “on /off-switch" mediated by exo+ polymerase is more reliable as compared to exo-polymerase in SNP assay and the novel “on/off” switch has enormous application in the diagnosis of monogenic diseases.