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目的建立一种准确、快速、高通量的apoE基因分型技术。方法从外周血样品提取基因组DNA,PCR扩增覆盖第112和158密码子的apoE基因片段;构建apoE基因片段重组质粒,并进行定点诱变,以得到3种等位基因型的对照样品;PCR产物消化处理,以除去残余的引物和dNTPs;进行模板指导的荧光染料标记终止碱基的掺入反应,应用荧光偏振检测仪分析荧光偏振值的变化;检测79例阿尔茨海默病(Alzheimer’sdisease,AD)患者和63名健康老年人的apoE基因型,分析基因型与AD易感性之间的关系。结果对分析结果进行测序验证,表明模板指导的荧光染料标记终止碱基掺入-荧光偏振检测技术分析结果与测序结果完全相符。AD组和健康对照组样品的基因分型结果提示apoEε4等位基因是迟发型AD的危险因素。结论应用此技术进行apoE基因多态性的基因分型分析,具有准确、简易和高通量等优势,可以作为AD风险分析的一种新技术,也适于apoE基因与其他疾病相关性研究时的大规模基因筛查分析。
Objective To establish an accurate, rapid and high-throughput apoE genotyping technique. Methods Genomic DNA was extracted from peripheral blood samples and the apoE gene fragment covering the 112th and 158th codons was amplified by PCR. The apoE gene fragment was constructed and subjected to site-directed mutagenesis to obtain control samples of three alleles. PCR The products were digested to remove residual primers and dNTPs; template-directed fluorescent dye labeling was used to stop the incorporation of bases, and the change of fluorescence polarization was analyzed by fluorescence polarization detector; 79 cases of Alzheimer’s disease sdisease (AD) patients and 63 healthy controls. The association between genotype and AD susceptibility was analyzed. Results The analysis of the results of sequencing validation, indicating that the template-guided fluorescent dye tag termination base incorporation - fluorescence polarization detection technology analysis results and sequencing results are fully consistent. Genotyping of samples from the AD and healthy controls suggested that the apoEε4 allele was a risk factor for late-onset AD. Conclusions Genotyping apoE gene polymorphism using this technique has the advantages of accuracy, simplicity and high throughput. It can be used as a new technique for AD risk analysis and is also suitable for the study of the correlation between apoE gene and other diseases Large-scale genetic screening analysis.