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[Purpose] To apply the "on/off switch of 3' exonuclease in single base discrimination of L191P and F275S mutations in gene KCNQ1 which are the mutations found in Chinese patients of LQTS. [Methods] A large amount of templates of wild type and mutation type were prepared by primer extension performed using polymerases without 3' exonuclease activity respectively. Allelic specific primers targeting wild type and mutation type templates were desgined with 3' terminal phosphorothioate modification. Two-directional primer extension was performed using polymerases with 3' exonuclease activity. [Results] Whether single PCR or multiplex PCR amplified by exo polymerase, allelic specific primers targeting wild type allele were extended while no products were generated from primers targeting point-mutated LQTS-related allele, and also allelic specific primers targeting point-mutated LQTS-related mutation type allele were extended while no products were generated from primers targeting wild type allele. [Conclusion] These data suggest that the "off-switch" mediated by exo polymerase is more reliable in the identification of LQTS mutation sites and the "on/off switch has potential enormous application in the diagnosis of monogenic diseases.