PCR-RFLP法检测亚甲基四氢叶酸还原酶基因C677T突变

C677T mutation of MTHFR gene detected by polymerase chain reaction-restriction fragment length polymorphism

目的 建立一种简便、实用的检测亚甲基四氢叶酸还原酶 (MTHFR)等位基因 C677T点突变的方法 ,并初步观察部分健康老人和老年血管性痴呆 (VD)患者中 MTHFR等位基因 C677T点突变情况。方法 PCR特异性扩增 MTHFR基因序列 ,扩增产物用限制性内切酶 Hinf 酶切 ,经聚丙烯酰胺凝胶电泳 (PAGE)分离、溴化乙锭染色后 ,观察酶切位点的限制性片段长度多态性 (RFLP)图谱。共检测标本 1 67例 ,其中健康老人(≥ 65岁 ) 1 38例、老年 VD 2 9例 ,并计算了各基因型频率和等位基因频率。结果 健康老人组和老年 VD患者组均检测到 C等位基因野生型 (CC)、杂合子 (CT)和 T等位基因纯合子 (TT)基因型 ,各组 MTHFR基因的 C677T点突变中 T突变位点的频率分别为 43.8%、51 .7%。结论 该方法简便、实用 ,适于一般实验室应用及流行病学调查。

Objective To establish a simple and practical method for detecting the MTHFR gene C677T mutation so as to investigate MTHFR genotypes in the healthy elder and Vascular Dementia (VD). Methods MTHFR allelic gene was amplified by polymerase chain reaction(PCR). PCR products were digested with HinflⅠ and subjected to PAGE and stained by EB. The patterns of restriction fragment length polymorphism (RFLP) of MTHFR allelic gene were distinguished. Total of 167 cases were investigated, including 138 cases healthy aged people(≥65 years old) and 29 cases VD. Results The genotypes of homozygous wild type CC / heterozygote CT / homozygote TT appeared in both groups. The frequencies of MTHFR C677T mutation in the healthy elder and VD group were 43.8% and 51.7%, respectively. Conclusions PCR-RFLP method is considered a simple and practical technique for obtaining accurate results in this research. This method is suitable for routine laboratories and epidemiology studies on the MTHFR polymorphism determination.