SNP敏感性分子开关对神经性耳聋GJB3中C→T突变点的识别

目的 利用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”系统识别神经性子GJB3中C→T突变点。方法 以非耳聋志愿者染色体DNA为模板，采用配对及三末端不配对的3’硫化修饰引物，使用不同保真度DNA聚合酶进行引物延伸反应。结果 该方法仅能使野生型基因相关的引物得以延伸，而耳聋基因相关引物则不能延伸。结论 本方法可在单碱基水平对遗传病相关基因进行特异性检测，提示其在单基因遗传病的诊断中具有广阔的应用前景。     

聚合酶3′外切活性对3′硫化修饰引物聚合反应的影响

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,SHIP)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。     

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

Objective:To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo polymerase, in single nucle- otide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods: We used the mtDNA 10400 locus to design unmodi- fied 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 exo-polymerase. Conclusion: The"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.