Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mt...Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled 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 exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified 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.展开更多
基金supported by a grant from the "135" Foundation of Jiangsu Province(RC2002052).
文摘Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled 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 exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified 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.
