Discrimination of mitochondrial DNA 10400 locus by SNP-operated on/off Switch

ObjectiveTo apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo+ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus.MethodsWe used the mtDNA 10400 locus to design unmodified 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.ResultsThe 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.ConclusionThe, “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.