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目的 建立一种简单、快速测定三磷酸腺苷结合盒转运子 1(ATP binding- cassettetransporter 1,ABC1)基因突变点 I82 3M的方法。方法 设计两种等位基因特异性引物 ,分别与 DNA双链的两条单链互补 ,其 3′端正好与单核苷酸多态性 (single nucleotide polymorphism ,SNP)位点重合 ,由引物的 3′端控制着引物的延伸反应 ,根据延伸反应的长度确定等位基因的类型 ,并通过在引物的 3′端区域引入一个人为不匹配碱基来提高延伸反应的特异性。结果 人为引入错配碱能提高单核苷酸多态性分析的特异性 ;通过优化实验条件 ,用本室建立的双等位基因特异性扩增法 ,可测定人 ABCA1基因突变点 I82 3M的不同 SNP类型。结论 人工修饰双等位基因特异性引物扩增法可用于人基因组中单核苷酸多态性的快速测定 ,不需使用复杂仪器设备 ,便于推广使用。
Objective To establish a simple and rapid method to determine the mutation point I82 3M of ATP binding-cassette transporter 1 (ABC1) gene. Methods Two allele-specific primers were designed, which were respectively complementary to two single strands of DNA double strand. The 3 ’end coincided with the single nucleotide polymorphism (SNP) The 3 ’end controls the extension reaction of the primer, determines the type of the allele according to the length of the extension reaction, and enhances the specificity of the extension reaction by introducing a human mismatch base in the 3’ end region of the primer. Results The introduction of mismatched alkaloid could improve the specificity of single nucleotide polymorphism analysis. By optimizing the experimental conditions and using the double-allele-specific amplification method established in this study, we can determine the mutation point of human ABCA1 gene I82 3M Different SNP types. Conclusion The artificial modified double allele-specific primer amplification method can be used for the rapid determination of single nucleotide polymorphisms in the human genome, without the use of complex equipment, easy to promote the use.