一种双脱氧核苷封闭的寡聚核苷酸制备方法

A method for synthesis of dideoxynucleotide blocked oligonucleotides

[目的]建立一种高效、简便制备双脱氧核苷(ddATP、ddTTP、ddGTP和ddCTP)封闭寡聚核苷酸的方法。[方法]选取乙醛脱氢酶(ALDH)基因,设计特异性上、下游引物。使用末端转移酶和碱性磷酸酶对引物进行双脱氧核苷修饰,利用荧光定量PCR对制备的封闭引物进行验证,并结合校读PCR对突变体进行检测。[结果]经过ddNTP封闭的引物,在普通荧光PCR体系中无法进行引物延伸;在校读PCR体系中则可以区分ALDH2的两种等位基因。[结论]该方法可以高效合成4种双脱氧核苷封闭的寡聚核苷酸,并且这种封闭的引物可以结合校读PCR对单核苷多态性进行检测。

[Objective] To develop a simple method for preparation of dideoxynucleotides（ddNTP）-blocked oligonucleotides.[Methods] Specific forward and reverse primers were designed according to ALDH2 gene.Terminal transferase was used to addddNTP onto the 3’end of the oligonucleotides and alkaline phosphatase was utilized to destroy redundant ddNTP.The obtained ddNTP-blocked primers were confirmed by fluorescence quantitative PCR,and further were applied to detect allelic mutations using Proof-Reading PCR（PR-PCR） with primers modified by dideoxynucleotide.[Results] The ddNTP-blocked primers were unable to initiate the primer extension in normal fluorescence PCR.In the PR-PCR,these blocked primers can well distinguish ALDH SNP.[Conclusion] The method could efficiently synthesize four kinds of ddNTP-blocked oligonucleotides,and the modified primers could be used to detect single nucleotide polymorphisms combining with PR-PCR.