CAJ | 학술논문

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。
설계합성료일충장비발협형핵산탐침,결합핵산외절매Ⅲ수해반응건립료일충면표기형광신호방대고령민검측DNA적신방법。당불존재파DNA시, SYBR Green Ⅰ형광염료능구감입발협형탐침적경부이발출흔강적형광,이당존재파DNA병여발협형탐침잡교후,핵산외절매Ⅲ종잡교산물적3’단개시수해발협형탐침,석방출파DNA,병촉발하일개매수해반응,동시SYBR Green Ⅰ염료야수발협형탐침수해이석방,도치형광신호강저,종이실현료대DNA적면표기형광신호방대고령민검측。해방법적검출한저지320 fmol/L,비전통쌍표적분자신표적방법강저료4~5개수량급,차해방법환구유면표기、간단、쾌속적특점。
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.