基于切割酶循环放大信号的双螺旋DNA检测

摘要：本论文基于金纳米粒子对荧光信号的猝灭结合酶循环信号放大实现双螺旋DNA的高灵敏检测。首先，设计了具有茎-环结构的发卡探针与目标双螺旋DNA杂交而形成三螺旋DNA结构作为辅助探针，打开的发卡探针相继与金纳米粒子标记的DNA探针杂交而形成双螺旋结构。由于荧光团与金纳米粒子的接近，而使荧光信号得以猝灭。然后，形成的双螺旋结构被切割酶切割，荧光探针得以释放，在此过程中，辅助探针的结构保持不变，重新与下一个金纳米粒子标记的探针杂交，实现信号的循环放大。在最佳实验条件下，双螺旋DNA的检测灵敏度可以降低到3.8pmol/L，同时我们的实验策略对双螺旋DNA检测具有很好的特异识别特性。51845

毕业论文关键词：金纳米粒子酶循环双螺旋DNA 荧光法

Detection of double-stranded DNA based on the loop amplified signal of cutting enzyme

Abstract：An ultrasensitive fluorescent platform for sequence-specific recognition of double stranded (dsDNA) based on the quenching of gold nanoparticle (AuNPs) to fluorophore labeled DNA probe was developed. A molecular beacon (MB) with “stem-loop” structure was introduced and hybridized with target dsDNA, inducing the formation of triplex DNA and opening the “stem-loop” structure of it, which was used as a helper probe (HP) to hybridize with fluorophore labeled DNA-AuNPs probe for the formation of double strand DNA. Because of the vicinity of fluorophore-modified DNA to the AuNPs surface, the fluorescence signal was efficiently quenched either in DNA-AuNPs probe or after the hybridization of such it with HP. Then, a double strand DNA formed based on the hybridization between DNA-AuNPs probe and HP could be recognized and cleaved by nicking enzyme, resulting in the release of fluorophore and the appearance of fluorescence signal. Because HP remaining intact during this process, it could be reused to hybridize again with the next DNA-AuNPs probe, triggering the happen of nicking nuclease assisted signal amplification. Under optimal conditions, a low detection limit of 3.8pmol/L was obtained for dsDNA detection, and the assay has high sequence specificity for dsDNA detection.

Keywords: gold nanoparticles,enzyme circulation,dsDNA,fluorescence method

前言 1

1 材料与方法 1

1.1 实验试剂 1

1.2 实验仪器 2

1.3 金纳米粒子的制备 2

1.4 检测探针修饰的金纳米粒子探针的制备 2

2 结果与分析 3

2.1 双螺旋DNA的检测原理 3

2.2 金纳米粒子和DNA-金纳米粒子探针的表征 3

2.3 双螺旋DNA的荧光法检测 4

2.4 实验条件的优化 5

2.5 目标双螺旋DNA的定量检测 6

3 讨论 7

致谢 7

参考文献 8

前言

人体中DNA都是以双螺旋DNA存在，并且在现在生命健康诊断中起着相当重要的作用。因此，双螺旋DNA的检测在医疗诊断和基因治疗中有相当重要的价值[1-3]。过去十年，相继发展了电化学、荧光、动态光和比色法来实现双螺旋DNA的高灵敏检测[4-10]。其中，荧光方法由于其良好的选择性、快速的分析过程和低的成本等优点受到了研究者们的广泛关注。然而，荧光分析方法的检测灵敏度往往比较低。因此，为了降低荧光检测的灵敏度，发展有效的信号放大策略是相当重要的。源`自·751.文/论:文'网,www.751com.cn 基于切割酶循环放大信号的双螺旋DNA检测:http://www.751com.cn/shengwu/lunwen_55618.html