MS-PCR法检测血管紧张素原基因M235T多态性

Application of MS PCR method to detect angiotensinogen gene M235T variants

目的 建立新型突变基因分离聚合酶链反应 (MS PCR)法检测血管紧张素原 (AgT)基因M 2 35T多态性 ,以正确评估其与家族性原发性高血压的关系。方法 建立PCR RFLP和MS PCR法最佳实验体系 ,验证敏感性及特异性。结果 ( 1)PCR RFLP体系中 ,随内切酶量和消化时间的增加 ,消化反应越彻底 ,以 0 0 75 μg基因组DNA扩增产物、30UnitTth111 Ⅰ酶消化 3小时为最佳反应体系。 ( 2 )MS PCR法一次扩增即可确定AgT基因型。 ( 3) 10个标本以PCR RFLP法检测者基因型均为MT ,而MS PCR法仅 1例为MT型 ,余 9例均为TT型 ( 90 % )。结论 传统的PCR RFLP可能因消化不彻底而造成结果判断的误差 ;MS PCR以其高敏感性和特异性成为检测基因多态性的快速、简便。

Objective Establish new mutagenically separated allele specific polymerase chain reaction(MS PCR) technique to detect the angiotensinogen gene M235T(AgT M235T) polymorphism and evaluate its relationship with essential familial hypertension.Methods Establish optimal systems of PCR RFLP and mutagenically separated allele specific polymerase chain reaction(MS PCR),then check their sensitivity and specificity.Results (1)0 075μg genomic DNA was digested by 30Unit Tth111 Ⅰ enzyme for three hours in PCR RFLP system.The more the enzyme used or the longer the digestive time, the more complete the reaction was.(2)Only one step PCR could determine the genotype in MS PCR system.(3)The genotype of ten samples were all showed MT types by PCR RFLP,however only one sample showed MT type,the remaining were TT type(90%),which were demonstrated by DNA sequencing.Conclusion The result of modified PCR RFLP was influenced by the incomplete digestion.Compared with PCR RFLP,MS PCR is a rapid,simple and reliable method to detect gene polymorphism.