SNP敏感性分子开关对神经性耳聋GJB3中C→T突变点的识别

Discrimination of C→T Point Mutation in GJB3 Senorineural Deafness by SNP-operated on/off Switch

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”系统识别神经性耳聋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 。

Objective To apply the 'on/off' switch of 3′ exonuclease in single base discrimination of C to T point mutation in GJB3 sensorineural deafness gene. Methods Two-directional primer extension was performed using polymerases with and without 3′ exonuclease activity. The 3′ phosphorothioate-modified allele- specific primers were evaluated in their effects on primer-extension with genomic DNA harboring wild type allele only. Results Amplified by exo + polymerase, primers targeting wild type allele were extended while no products were generated from primers targeting point-mutated deafness-related allele. As a control, exo - polymerase yielded products from both types of primers. Conclusion These data suggest that the 'off-switch' mediated by exo + polymerase is more reliable as compared to exo - polymerase in SNP assay and the novel 'on/off' switch has enormous application in the diagnosis of monogenic diseases.