基于免标记发夹型探针和核酸外切酶Ⅲ的荧光信号放大DNA检测  

Label-free Fluorescence Signal Amplifying Detection of DNA Based on Hairpin Probe and Exo Ⅲ

作　　者：郭秋平[1,2,3] 赵下雨[1,2,3] 谢琴[1,2,3] 王柯敏[1,2,4,3] 万俊[1,2,3] 袁宝银 谭誉宇[2,4,3] 

机构地区：[1]湖南大学生物学院,长沙410082 [2]湖南大学化学生物传感与计量学国家重点实验室,长沙410082 [3]湖南大学生物纳米与分子工程湖南省重点实验室,长沙410082 [4]湖南大学化学化工学院,长沙410082

出　　处：《高等学校化学学报》2014年第8期1646-1651,共6页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金重大项目(批准号:21190044)、国家自然科学基金(批准号:21175035);国家“九七三”计划项目(批准号:2011CB911002);科技部国际合作专项(批准号:2010DFB30300);湖南省科学技术项目(批准号:2013FJ4042)资助~~

摘　　要：设计合成了一种长臂发夹型核酸探针,结合核酸外切酶Ⅲ水解反应建立了一种免标记荧光信号放大高灵敏检测DNA的新方法。当不存在靶DNA时, SYBR Green Ⅰ荧光染料能够嵌入发夹型探针的茎部而发出很强的荧光,而当存在靶DNA并与发夹型探针杂交后,核酸外切酶Ⅲ从杂交产物的3’端开始水解发夹型探针,释放出靶DNA,并触发下一个酶水解反应,同时SYBR Green Ⅰ染料也随发夹型探针水解而释放,导致荧光信号降低,从而实现了对DNA的免标记荧光信号放大高灵敏检测。该方法的检出限低至320 fmol/L,比传统双标的分子信标的方法降低了4~5个数量级,且该方法还具有免标记、简单、快速的特点。A novel label-free fluorescence signal amplifying method for DNA detection was developed with high specificity and sensitivity based on a long arm hairpin nucleic acid probe and exonuclease Ⅲ（ Exo Ⅲ） . Without the target DNA, the SYBR Green Ⅰ dye could be embedded into the stem of hairpin nucleic acid probe to generate strong fluorescence. While in the presence of target DNA, Exo Ⅲ could catalyze the step-wise removal of mononucleotides from 3’-OH termini of double-stranded DNA with the degradation of the hair-pin probe. After that, the target DNA was released, triggering the next cycle of exonuclease digestion reac-tion. Then the SYBR Green Ⅰ was continuously released, resulting in fluorescence intensity decreased. It rea-lized the label-free signal amplifying detection of DNA. The detection limit of this method was as low as 320 fmol/L. It can be expected to provide a novel, simple, label-free and rapid tool for DNA detection.

关 键 词：发夹型探针 核酸外切酶Ⅲ 免标记 信号放大 DNA检测 

分 类 号：O657.3[理学—分析化学]