错配杂交化学发光法检测亚甲基四氢叶酸还原酶基因多态性

Detection of methylenetetrahydrofolate reductase gene polymorphisms by chemiluminescence based on mismatch hybridization

目的建立一种基于错配杂交及化学发光技术的检测亚甲基四氢叶酸还原酶(m ethylenetetrahydrofolate reducatase,MTH-FR)基因多态性的新方法。方法针对多态位点设计两条寡核苷酸探针,分别与野生型及突变型序列互补。将两条探针分别与含多态位点C677T的PCR扩增产物杂交,然后用化学发光法检测,根据两条探针的发光值之比确定样品的基因型。结果用本法随机检测了50例DNA样品,结果3种基因型的发光值之比可以严格区分,其中野生型(C/C)15例,杂合型(C/T)28例,突变型(T/T)7例,与参考方法(PCR-RFLP)的检测结果完全一致。结论本方法操作简便、检测快速而且费用低廉,可广泛应用于MTHFR基因突变及多态性检测。

Objective To establish a new method for rapid detection of polymorphisms of methylenetetrahydrofolate reductase gene by chemiluminescence based on mismatch hybridization. Methods Two oligonucleotides probes for MTHFR polymorphic site were designed which perfectly match the wild-type and mutant genotype of C677T site,respectively. After hybridization with amplified DNA fragment, the hybrids were detected by a chemiluminescence method and the genotypes were identified by calculating the ratio of luminescence value obtained from the two probes. Results The polymorphisms of 50 samples were analyzed by the method. Wild-type, heterozygous and homozygous were detected in 15 cases,28 cases and 7 cases respectively. The genotypes of the 50 samples were accordant with the results detected by PCR-RFLP. Conclusion The established method is simple and rapid, and can be widely used for analysis of gene mutation.