Objective:To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo+ polymerase, in single nucle-otide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods: We used the mtDNA 10400 locus to design unmodi-fied and 3′phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3′exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results: The unmodified primers were extended by both exo- and exo+ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3′phosphorothioate-modified primers with a terminal mismatch triggered an “off-switch”of exo+ polymerase when compared to exo-polymerase. Conclusion: The“on/off”switch constituted by the combination of 3′phosphorothioate-modified primers with exo+ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.