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

doi:10.7503/cjcu20140321

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (8): 1646.doi: 10.7503/cjcu20140321

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

郭秋平^1,^2,⁴, 赵下雨^1,^2,⁴, 谢琴^1,^2,⁴, 王柯敏^1,^2,^3,⁴(), 万俊^1,^2,⁴, 袁宝银^2,^3,⁴, 谭誉宇^2,^3,⁴

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

收稿日期:2014-04-04 出版日期:2014-08-10 发布日期:2014-06-23
作者简介:联系人简介: 王柯敏, 男, 博士, 教授, 博士生导师, 主要从事化学生物传感技术及纳米尺度和分子水平上获取生物化学信息的研究. E-mail: kmwang@hnu.cn
基金资助:
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)和湖南省科学技术项目(批准号: 2013FJ4042)资助

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

GUO Qiuping^1,^2,⁴, ZHAO Xiayu^1,^2,⁴, XIE Qin^1,^2,⁴, WANG Kemin^1,^2,^3,^4,^*(), WAN Jun^1,^2,⁴, YUAN Baoyin^2,^3,⁴, TAN Yuyu^2,^3,⁴

1. College of Biology
2. State Key Laboratory of Chemo/Biosensing and Chemometrics
3. College of Chemistry and Chemical Engineering
4. Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Hunan University, Changsha 410082, China

Received:2014-04-04 Online:2014-08-10 Published:2014-06-23
Contact: WANG Kemin E-mail:kmwang@hnu.cn
Supported by:
Supported by the Major Program of the National Natural Science Foundation of China(No.21190044), the National Natural Science Foundation of China(No.21175035), the National Basic Research Program of China(No.2011CB911002), the International Science & Technology Cooperation Program of China(No.2010DFB30300) and Hunan Provincial Science and Technology Project of China(No.2013FJ4042)

摘要/Abstract

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.

关键词: 发夹型探针, 核酸外切酶 Ⅲ, 免标记, 信号放大, DNA检测

Abstract:

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 stepwise removal of mononucleotides from 3'-OH termini of double-stranded DNA with the degradation of the hairpin probe. After that, the target DNA was released, triggering the next cycle of exonuclease digestion reaction. 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.

Key words: Hairpin probe, Exo Ⅲ, Label-free, Signal amplification, DNA detection

中图分类号:

O657.3

TrendMD:

郭秋平, 赵下雨, 谢琴, 王柯敏, 万俊, 袁宝银, 谭誉宇. 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. 高等学校化学学报, 2014, 35(8): 1646.

GUO Qiuping, ZHAO Xiayu, XIE Qin, WANG Kemin, WAN Jun, YUAN Baoyin, TAN Yuyu. Label-free Fluorescence Signal Amplifying Detection of DNA Based on Hairpin Probe and Exo Ⅲ^†. Chem. J. Chinese Universities, 2014, 35(8): 1646.

图/表 8

Fig.1 Principle of label-free fluorescence amplifying detection of DNA based on hairpin probe and Exo Ⅲ

Fig.2 Feasibility of label-free fluorescence amplifying detection of DNA based on hairpin probe and Exo Ⅲ a. Adding 5 nmol/L target DNA; b. without target DNA; c. adding 5 nmol/L random DNA.

Fig.3 Agarose gel electrophoresis image lanes (A) Hairpin probe; (B) target; (C) hairpin probe with Exo Ⅲ reaction 30 min; (D) hairpin probe and target; (E) hairpin probe, target and Exo Ⅲ reaction 5 min; (F) hairpin probe, target and Exo Ⅲ reaction 30 min.

Fig.4 Influence of Exo Ⅲ concentration to DNA detection

Fig.5 Influence of probe concentration to DNA detection

Fig.6 Influence of SYBR Green Ⅰ concentration to DNA detection

Fig.7 Fluorescence curves response to the different concentrations of DNA(A) and dependence of fluorescence peak intensity at 530 nm on target concentration from 0—5000 pmol/L(B) (A) Concentration of DNA from a—h/(pmol·L-1): 0, 0.32, 1.6, 8, 40, 200, 1000, 5000.

Fig.8 Exo-amplified assay between perfectly matched and mismatched DNA targets The data shown were collected at 1 nmol/L target concentrations. a. Without target DNA; b. adding 5 nmol/L single-base mismatched DNA; c. adding 5 nmol/L target DNA.

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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^†

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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†

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