聚合酶3′外切活性对3′硫化修饰引物聚合反应的影响  

Effect of 3′ exonuclease activity of polymerase on extension of phosphorothioate-modified primers

作　　者：郭紫芬[1] 陈琳玲[1] 张佳 彭翠英[1] 杨向东[3] 张旭 何淑雅[3] 廖端芳[1] 李凯[1] 

机构地区：[1]南华大学药物药理研究所,衡阳421001 [2]津脉公司 [3]南华大学生物化学与分子生物学研究所,衡阳421001

出　　处：《中华医学遗传学杂志》2003年第4期328-330,共3页Chinese Journal of Medical Genetics

基　　金：国家"973"项目部分资助 (G2 0 0 0 0 5690 5)~~

摘　　要：目的　探讨硫化修饰的次 3′末端不配对引物能否引发高保真 DNA聚合酶介导的聚合反应的非成熟性终止 ,即所谓聚合反应的“关”效应。方法　采用配对及非末端不配对的 3′硫化修饰引物 ,研究其对不同保真度 DNA聚合酶引物延伸反应的影响。结果　非末端不配对的 3′硫化修饰引物也能引发高保真 DNA聚合酶介导的聚合反应非成熟性终止 ,而对低保真 DNA聚合酶所介导的聚合反应则无明显影响。同时 ,3′硫化修饰的配对引物对不同保真度 DNA聚合酶引物延伸反应均无影响。结论　硫化修饰的次 3′末端不配对引物与 3′末端不配对引物对引物延伸的影响相似 ,同样能引起高保真 DNA聚合酶介导的聚合反应的“关”的效应。显然 ,在单基因遗传病的诊断及单核苷酸多态性 (single nucleotide polymorphism,SNP)的高通量分析等方面 ,硫化修饰的碱基特异性引物与高保真 DNA聚合酶所构成的对 SNP敏感的“开 /关”系统 ,具有广阔的应用前景。Objective: To determine whether 3′phosphorothioate-modified -2 terminal mismatched primers can turn off DNA polymerization mediated by Exo + polymerase. Methods: Two-directional primer extension was performed using polymerase with and without 3′ exonuclease activity. The effects of unmodified primers and 3′ phosphorothioate-modified primers on primer extension were evaluated. Results: Exo- polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3′ phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported 'off-switch' of Exo+ polymerase. Conclusion: These data suggested that the 'off-switch' can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.

关 键 词：单核酸多态性 高保真聚合酶 硫化修饰引物 DNA 聚合反应 单基因遗传病 诊断 

分 类 号：R346[医药卫生—基础医学]