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

摘要

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.%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。