摘要
目的探讨硫化修饰的碱基特异性引物与高保真DNA聚合酶所构成的对单核苷酸多态性敏感的分子开关系统在基因组单核苷酸多态性检测中的特异性。方法以人类基因组DNA为模板,采用3’末端配对及不配对的3’末端硫化修饰引物,进行不同保真度DNA聚合酶介导的引物延伸反应。结果Taq酶介导的碱基特异引物延伸反应扩增产物出现非特异性带,而单核苷酸多态性敏感分子开关介导的引物延伸反应未出现非特异性带。结论单核苷酸多态性敏感分子开关是一种特异性强,可靠性高的可用于基因组单核苷酸多态性分析的新方法,可在单碱基水平对遗传病相关基因进行特异性检测。
Aim To analyze the specificity of the on/off switch mediated by high fidelity prootreadmg DNA polymerases and 3'-phosphorothioate-modified allele-specific primers for screening certain sites of single nucleotide polymorphism (SNP) in genomic DNA. Methods The genomic DNA is used as the template. The 3'-phosphorothioate-modified allele-specific primers were evaluated in their effects on primer-extension with genomic DNA harboring SNP sites allele. Results The products from Taq had many mixed bands , and some of them clearly showed false positives. However , at most of the situations , high fidelity DNA polymemse of Pfu yielded single products. Conclusion These data suggest that the “on/off-switch”mediated by exo+ polymerase is more reliable as compared to exo-polymemse in SNP assay and the novel “on/off ”switch has enormous application in the diagnosis of monogenic diseases.
出处
《中国动脉硬化杂志》
CAS
CSCD
2005年第3期279-281,共3页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(30171084)资助
国家973项目(G2000056905)部分资助