儿茶酚O-甲基转移酶基因多态性分析方法探讨  

Exploration of the approaches of analyzing the genetic polymorphism of catechol-O-methyltransferase

作　　者：赵晓苗[1] 赵一凡[2] 赵宇正[3] 赵炜疆 唐雪莲 谢梅青[1] 

机构地区：[1]中山大学孙逸仙纪念医院妇产科,广东广州510120 [2]中山大学孙逸仙纪念医院麻醉科,广东广州510120 [3]肇庆市第一人民医院 [4]汕头医学院神经科学中心 [5]深圳市妇儿医院

出　　处：《中国实验诊断学》2012年第11期2021-2024,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金(81100402);广东省自然科学基金(S2011040002767);广州市科技项目(2010Y1-C431)

摘　　要：目的建立限制性片段长度多态性(RFLP)和变性高效液相色谱(DHPLC)分析儿茶酚O-甲基转移酶(COMT)基因多态性的方法,探讨分析基因多态性与临床疾病发生风险关系的方法学。方法从子宫内膜癌患者和对照组的全血和蜡块包埋组织提取DNA,应用RFLP和DHPLC法检测COMT基因第4号外显子第158位密码子G/A的多态性,并结合DNA测序法分析两种方法的一致性。结果全血和蜡块组织提取的DNA均可扩增PCR产物,且可行酶切反应;所有全血和小部分蜡块DNA PCR产物可行DHPLC分析。测序法与RFLP法比较,Kappa值=0.697;与DHPLC测序法比较,Kappa值=0.924。结论 RFLP与测序法的一致性好,适用于全血和蜡块提取的DNA的基因多态性分析;DHPLC与测序法的一致性极好,但对机器和样本要求较高,适用于血DNA的基因多态性分析。Objective To set up the approaches of analyzing the genetic polymorphisms of Catechol-O-methyltransferase（COMT） with restriction fragment length polymorphism（RFLP） and denaturing High Performance Liquid Chromatography（DHPLC） and explore the technology of analyzing the relations between genetic polymorphisms and the susceptible risk of clinical disease.Methods After Abstracting DNA from whole blood and investing tissue of paraffin block,Polymerase-chain reaction（PCR）-RFLP and DHPLC were used to study the allele and genotype frequency distributions of COMT polymorphism Val158Met among 50 endometrial cancer patients and 50 control women.And DNA sequence analysis was combined to compare the consistency of the two methods.Results PCR and RFLP were done successfully from both blood and paraffin block DNA.And all the PCR products from blood DNA but only minor part from paraffin block DNA can be analyzed by DHPLC.When the DNA sequence analysis results were compared with RFLP results,Kappa value was 0.697,but when compared with DHPLC results,Kappa value was 0.924.Conclusion The consistency between RFLP and DNA sequence analysis was good,and RFLP method was fit to the gene polymorphism analysis of the DNA extracting from both blood and paraffin block.Meanwhile,the consistency between DHPLC was extremely good,but DHPLC demanded highly on the machine and sample,suitable for the gene polymorphism analysis of the DNA extracting from blood.

关 键 词：单核苷酸多态性 限制性酶切片段长度 变性高效液相色谱 儿茶酚O-甲基转移酶 子宫内膜癌 

分 类 号：R440[医药卫生—诊断学]