PCR结合银染法检测载脂蛋白E等位基因

PCR IN COMBINATION WITH SILVER STAINING AS A METHOD FOR THE DETERMINATION OF APOLIPOPROTEIN E ALLELES

目的介绍一种高效、灵敏、安全的载脂蛋白Ｅ等位基因检测法。方法采用非酚法快速提取人全血基因组ＤＮＡ，ＰＣＲ扩增包含两个等位基因决定位点的ＤＮＡ片段，扩增产物经限制性酶切后进行聚丙烯酰胺凝胶电泳，用简易银染法显示酶切片段。结果银染显示出清晰易辨的ＤＮＡ酶切小片段，整个检测过程２４小时完成。将此法初步用于１９例散发性阿尔茨海默病患者和４１名无精神神经疾患老年人载脂蛋白Ｅ等位基因的检测，测得ε４等位基因频率分别为０．２９和０．０２，两者之间差异非常显著（Ｐ＜０．００１）。结论ＰＣＲ结合银染法检测载脂蛋白Ｅ等位基因，在尽量避免使用有害试剂的同时，提高了检测灵敏度和效率。

Objective To present a rapid, sensitive and safe procedure for the determination of human apolipoprotein E alleles.Methods Genomic DNA was extracted from 0.5ml whole blood by a non phenol protocol. After PCR and restriction enzyme digestion,short DNA fragments were detected by a simplified silver staining method. This technique was used to assay apolipoprotein E alleles in 19 patients with sporadic Alzheimer's disease and 41 normal aged persons. Results The short DNA fragments were visualized clearly on polyacrylamide gel processed by silver staining. It took 24 hours from DNA extraction to the final result. The ε4 allele frequency in patients with sporadic Alzheimer's disease was 0.29, much higher than that in control group (0.02, P<0.001) . Conclusion PCR in combination with silver staining can satisfy the need for high resolution and high effciency in the determination of apolipoprotein E alleles and can be used as a routine procedure in clinical and epidemiological investigations.