高通量apoE基因分型技术的建立及应用

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’s disease，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。

A rapid and high throughput method for apoE genotyping

OBJECTIVE: To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput. METHODS: Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated. RESULTS: The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD. CONCLUSION: TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.