错配杂交酶联免疫法检测亚甲基四氢叶酸还原酶基因多态性

Detection of methylenetrtrahydrofolate reductase polymorphisms with a colorimetric method based on mismatch hybridization

目的建立一种基于错配杂交及酶联免疫检测亚甲基四氢叶酸还原酶(Methylenetetrahy drofolate redu-catase,MTHFR)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。将两条地高辛标记的探针分别与含多态位点C677T的PCR扩增产物杂交,然后显色检测,根据两条探针的吸光度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,结果可见三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。

Objectlve To establish a new method for rapid detection polymorphisms of methylenetrtrahydrofolate reductase with a colorimetric method 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 with a colorimetric method and the genotype were identiffed by calculating the ratio of absorbency obtained with two probes. Results The polymorphism of 50 samples were analysed by the method. Wild-type, heterozygous and homozygous were detected in 15 cases, 28 cases and 7 cases, respectively. Genotypes of the 50 samples is accordance with the results detected by PCR-RFLP.Condusion This method is simple and rapid,and can be widely used for analysis gene mutation.