Discrimination of KCNQ1 point mutations in long QT syndrome by on/off switch

[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.