基于高区别因子自组装三臂DNA纳米探针检测人体多重耐药基因  

High Discrimination Factor Self-Assembly Three-Arm DNA Nanoprobe for Detection of Human Multidrug Resistance Gene

作　　者：许榜田 杨晓兰[2] 白书连 赵语 XU Bang-tian;YANG Xiao-lan;BAI Shu-lian;ZHAO Yu(University-Town Hospital of Chongqing Medical University, Chongqing 401331;Department of Laboratory Medicine ,Chongqing Medical University ,Chongqing 400016)

机构地区：[1]重庆医科大学附属大学城医院,重庆401331 [2]重庆医科大学检验医学院,重庆400016

出　　处：《分析科学学报》2018年第3期303-309,共7页Journal of Analytical Science

基　　金：国家自然科学基金(No.31570862)

摘　　要：建立了一种新的基于高区别因子三臂DNA纳米探针的荧光分析法检测人体多重耐药(MDR)基因。此探针由保护链P1和互补链C1与信号报告部分(信号链S1和信号链S2)组成:P1与修饰了荧光猝灭基团Dabcyl的S1及C1部分碱基杂交;修饰了荧光基团FAM的S2与C1另一部分碱基杂交。探针内部Dabcyl和FAM相互靠近,荧光猝灭。当MDR基因存在时,其与三臂DNA纳米探针发生链替代反应,释放荧光信号。在最优条件下,单碱基错配产生的微小的热力学改变即可影响该探针链替代效率,从而实现高特异性检测MDR基因和其点突变基因(2m、3m、3d、5m、5d)。该探针对不同突变位点及同一突变位点的不同突变类型MDR基因均展现出良好的特异性,其检测3d的区别因子达到24.1,平均区别因子达到11.8。在混合人血清中,MDR基因的回收率为99.0%~101.7%。此外,本方法通用性好,不需要重新设计S1和S2即可实现对miR-21的特异性检测。A novel fluorometric method for detecting multidrug resistance（MDR）genes in human with high discrimination factor was developed based on three-arm DNA nanoprobe.The probe consisted of a protection DNA strand P1,a complementary DNA strand C1 and a signal reporting part（signal strand S1 and signal strand S2）.P1 could hybridize with Dabcyl modified S1 and part bases of C1.FAM modified S2 could hybridize with another part of bases of C1.Dabcyl and FAM were close to each other inside the probe,and the fluorescence was quenched.In the presence of MDR gene,it could displace P1 from three-armed DNA nanoprobe,which separated Dabcyl and FAM,thus releasing an intensive fluorescence signal.Under the optimal condition,a slight thermodynamic change caused by single-base mismatch MDR could affect the efficiency of strand displacement reaction,therefore high specificity detection of the MDR gene and its point mutation genes（2 m,3 m,3 d,5 m,5 d）was achieved.The probe showed good specificity for MDR genes with different mutation sites and different mutation types at the same mutation site.Discrimination factor of the probe was 24.1 for 3 ddetection.Mean discrimination factor was 11.8 for all the mutant MDR genes detection.In mixed serum,the MDR gene recovery was 99.0%-101.7%.In addition,the method had good universality and could realize the specific detection of miR-21 without redesigning S1 and S2.

关 键 词：DNA探针 荧光分析法 多重耐药基因 特异性 点突变 

分 类 号：O657.39[理学—分析化学]