纳米磁珠法与试剂盒法分析载脂蛋白E基因的多态性

摘要

背景：相对于血液标本，口腔拭子标本更利于大规模载脂蛋白E基因多态性分析的研究，但目前对口腔拭子标本基因组DNA的提取尚无统一方法。　　目的：探索合适的口腔拭子标本基因组提取方法以分析载脂蛋白E基因的多态性。　　方法：收集50例散发性阿尔茨海默病患者口腔拭子标本，每份标本分别应用纳米磁珠法与PicoDNA微量核酸提取试剂盒法提取基因组DNA，对比分析两种方法获得的基因组DNA纯度和浓度，后续进行PCR反应，应用DNA电泳确认有无成功扩增出目的条带，通过DNA测序方法分析载脂蛋白E基因的多态性。　　结果与结论：两种方法提取的基因组DNA纯度均较好，纳米磁珠法提取的基因组DNA浓度要高于PcioDNA微量核酸提取试剂盒法所得浓度(P<0.05)。两组获取的基因组DNA均可成功进行PCR扩增，但电泳结果示纳米磁珠法扩增的目的条带更清楚。两组PCR产物DNA测序结果一致，载脂蛋白E基因ε2、ε3、ε4基因型的比例分别是6%，71%，23%。表明纳米磁珠法相对于PcioDNA微量核酸提取试剂盒法提取口腔拭子标本基因组DNA更适合用于大样本载脂蛋白E基因多态性的研究。%BACKGROUND:Relative to blood samples, mouth swab samples are more beneficial for apolipoprotein E gene polymorphism analysis among large cohorts. However, agreement has not yet been reached about how to extract genomic DNA form mouth swab samples. OBJECTIVE:To develop an appropriate method to extract genomic DNA form mouth swab samples, which are suitable for apolipoprotein E gene polymorphism analysis. METHODS:Fifty mouth swab samples from patients with sporadic Alzheimer’s disease were col ected. Magnetic nanoparticles and PicoDNA trace nucleic acid extraction kit were used to extract genomic DNA form mouth swab samples. And the purity and concentration of the genomic DNA extracted by the two methods were analyzed. Then PCR amplifications and DNA electrophoresis were performed to confirm whether the genomic DNA was able to amplify desired DNA fragments. DNA sequencing was applied to analyze apolipoprotein E gene polymorphisms. RESULTS AND CONCLUSION:Genomic DNA extracted by the two methods was of high purity. The concentration of genomic DNA extracted by magnetic nanoparticles was higher than by PicoDNA trace nucleic acid extraction kit, and the difference had statistical significance (P<0.05). Al the genomic DNA were able to performed PCR amplifications to obtain desired PCR products, but results of DNA electrophoresis showed that DNA fragments were more clear by nanoparticles method. The results of DNA sequencing were the same by the two methods. The distribution ofε2,ε3,ε4 genotypes of apolipoprotein E gene was 6%, 71%, 23%, respectively. Magnetic nanoparticles were better than PicoDNA trace nucleic acid extraction kit for extracting genomic DNA form mouth swab samples for apolipoprotein E gene polymorphism analysis.