中文摘要：

目的通过改进和优化多重扩增阻滞突变系统-PCR（multi-ARMS-PCR）条件,建立载脂蛋白E（ApoE）基因的简易分型方法。方法基于multi-ARMS-PCR的原理和特点,针对文献报道方法中存在的缺陷和错误,重新设计或改进引物。以外周血白细胞基因组DNA为模板,应用4个等位基因特异性寡核酸上游引物、1个通用下游引物和一对内参引物,分A,B两个管同步进行多重PCR反应。PCR扩增产物经过琼脂糖凝胶电泳分离-EB染色,根据电泳带型的差异,实现对ApoE 6种基因型的判定。结果新引物显著提高了扩增效率和反应特异性,排除了非特异条带的干扰,减少了ApoE基因分型的错判。结论采用优化后的multi-ARMS-PCR方法对ApoE基因型进行鉴定,具有操作简便、时间短、效率高、成本低的优点,值得推广。

英文摘要：

Objective To set up a simple method for apolipoprotein E （ApoE） genotyping by modifying the conditions of multiplex amplification refractory mutation system PCR （multi-ARMS-PCR）. Methods Based on the principle of multi-ARMS-PCR and considering the faults existed in some published papers, new primers were designed to improve PCR conditions. DNA genome of peripheral white blood cells was used as the template. Four allele-specific oligonucleotide upstream primers, one common downstream primer and a pair of internal positive control primers were constructed. Multi-ARMS-PCR were performed with combination of different primers in 2 reaction tubes synchronously. Amplified multiplex products were electrophoresed on agarose gels containing ethidium bromide. Six ApoE genotypes were distinguished by the band sizes. Results Using the new primers, amplification efficiency and specificity were significantly increased and the misclassification was diminished due to removing the interference of non-specific bands. Conclusion The optimized multi-ARMS-PCR is an easy, time-saving, efficient and economical method for determination of ApoE genotypes applied in a minimally equipped laboratory.