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
Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by fi...Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes.This unusual DNA modification,called phosphorothioation,is the first physiological modification identified on the DNA backbone,in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner.Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community.Here,we summarize the progress in chemical,genetic,enzymatic,bioinformatical and analytical aspects of this novel postreplicative DNA modification.We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.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.
文摘Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes.This unusual DNA modification,called phosphorothioation,is the first physiological modification identified on the DNA backbone,in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner.Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community.Here,we summarize the progress in chemical,genetic,enzymatic,bioinformatical and analytical aspects of this novel postreplicative DNA modification.We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.
